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Les malalties neurodegeneratives es desenvolupen gràcies a diferents mecanismes moleculars, que produeixen agregació de proteïnes mal plegades en diverses àrees cerebrals. D'una banda, l'α-sinucleïna es troba als cossos de Lewy presents a la malaltia de Parkinson i a les demències amb cossos de Lewy (DCL: DCL de tipus pur, DCLp; DCL de tipus comuna, DCLc) i a les inclusions citoplasmàtiques glials, que són la característica neuropatològica de l'atròfia multisistèmica (AMS). D'altra banda, les plaques senils formades principalment per β-amiloide i cabdells neurofibril·lars amb tau hiperfosforilada són la peculiaritat principal de la malaltia d'Alzheimer (MA). A més, la MA es caracteritza per un dèficit colinèrgic, el qual és encara més acusat a les DCL. En el present treball s'ha estudiat la genètica de la butirilcolinesterasa (BChE), un enzim de tipus glicoproteic que intervé en el metabolisme de l'acetilcolina, sobre les sinucleïnopaties en comparació amb la MA com a control de neurodegeneració, així com mostres control. L'estudi es va iniciar analitzant la freqüència de la variant K (A1699G, A539T) de BChE que està descrit com un possible factor de risc a patir la MA a les mostres post mortem (25 MA, 12 DCLp, 24 DCLc, 5 MP, 6 AMS i 25 controls). Es va observar una marcada acumulació de l'al·lel K de BChE en totes les demències en comparació amb els controls. A continuació es va caracteritzar el promotor de BChE mitjançant seqüenciació i s'hi van trobar quatre polimorfismes no descrits. Aquests es van genotipar a totes les mostres post mortem i els resultats van ser analitzats conjuntament per identificar possibles combinacions genotípiques específiques formades pels polimorfismes del promotor i la variant K de BChE. El resultat més rellevant va ser la detecció d'una combinació genotípica, AAAGCC8+K+, específica per al 17% de les mostres amb DCL. L'aplicació del genotipat de BChE com a eina per al diagnòstic diferencial augmentaria l'especificitat diagnòstica de les DCL, que tot i que l'any 2005 es van establir nous criteris per al seu diagnòstic clínic, es diagnostiquen erròniament entre el 40 i el 80 % dels casos. Per corroborar les dades obtingudes a partir de mostres post mortem, es va fer un estudi a partir de mostres de sang de 230 pacients amb MA, 21 amb AMS, 23 amb MP i 160 controls. Per completar l'anàlisi de BChE, es va analitzar l'expressió relativa de les quatre variants de splicing a nivell d'mRNA al còrtex frontal, còrtex temporal, nucli caudat i cerebel utilitzant PCR a temps real. El resultat va ser l'obtenció de patrons d'expressió determinats per a cada malaltia estudiada. Finalment, per immunohistoquímica es va concloure que BChE no es troba en els cossos de Lewy, ja que no colocalitza amb α-sinucleïna. Encara que per a la MA s'han descrit diferents marcadors genètics, fins al moment no se'n coneix cap per a les DCL. Mentre que els patrons d'expressió de mRNA característics de cada patologia trobats poden ajudar a elucidar els possibles mecanismes moleculars involucrats en el desenvolupament de les DCL, la determinació de les combinacions genotípiques específiques podran ajudar en el seu diagnòstic diferencial. / Neurodegenerative diseases develop through different molecular mechanisms that produce aggregation of unfolded proteins in different brain areas. On one hand, α-synuclein is present in Lewy bodies that can be found in Parkinson's disease and in dementia with Lewy bodies (DLB: pure DLB, pDLB; common DLB, cDLB) and in glial cytoplasmic inclusions (GCI) that are the pathological hallmark of multiple system atrophy (MSA). On the other hand, senile plaques composed primarily of β-amyloid and neurofibrillary tangles with hyperphosphorilated tau are the main characteristic of Alzheimer's disease (AD). In addition, AD is characterized by a cholinergic deficit, which is even more pronounced in DLB. In this thesis we have studied Butyrylcholinesterase (BChE), a glycoproteic enzyme that mediates acetylcholine metabolism, in synucleinopathies compared with AD, as a control of neurodegeneration, as well as, with control samples.The study began by analysing the frequency of BChE K-variant (A1699G, A539T), that is described as a possible risk factor to suffer AD, in post mortem samples (25 AD, 12 pDLB , 24 cDLB, 5 PD, 6 MSA and 25 controls). There was a marked accumulation of BChE K allele in all dementias compared with controls.Then BChE promoter was characterized and four new polymorphisms were found. These were genotyped in all post mortem samples, results were analyzed together to identify specific genotypic combinations formed by polymorphisms of the promoter and BChE K-variant. The most relevant result was the detection of a genotypic combination, AAAGCC8+K+, specific for 17% of DLB samples. The application of BChE genotyping as a tool for differential diagnosis would increase the diagnostic specificity of DLB, that although in 2005 new criteria for its clinical diagnosis were established, between 40-80% cases are misdiagnosed. To corroborate the data obtained from post mortem samples, a study from blood samples of 230 AD patients, 21 with MSA, 23 with PD and 160 controls was done.To complete the analysis of BChE, we examined the relative expression of four splice variants in frontal cortex, temporal cortex, caudate nucleus and cerebellum using real time PCR. Specific expression patterns for each disease studied were obtained. Finally, immunohistochemistry concluded that BChE is not found in Lewy bodies, as it does not colocalize with α-synuclein.Although several AD genetic markers have been described, until this moment none is known for DLB. While the mRNA expression patterns characteristic of the disease may help to elucidate the possible molecular mechanisms involved in the development of DLB, the determination of the specific genotypic combination can help in their differential diagnosis.

α-synuclein is a small presynaptic protein whose misfolding and aggregation are considered drivers of the neurological disorders Parkinson’s disease, multiple system atrophy, dementia with Lewy bodies and related synucleopathies. α-synuclein exists in a dynamic state that changes from an α-helical conformation when bound to liposomes to natively unfolded in solution, the majority being in the latter state. The disease process by which native healthy α-synuclein undergoes a change in conformation to form β-sheet oligomers and fibrils is still unresolved. The fibrillation process has been widely studied by several different techniques and the structure of the fibrils has been determined by NMR, scanning transmission electron microscopy and X-ray diffraction. The early stages of aggregation into β-sheet rich oligomers, despite having been widely studied, has proven difficult to follow due to the heterogeneity of the species formed and the unpredictability of the process. The goal of the work reported here was to design and develop a novel, reproducible and quantitative assay to study the early stages of α-synuclein aggregation and to establish a platform for discovery of novel compounds that inhibit this process. These compounds could then be taken as a starting point for the development of new drugs for the treatment of synucleopathies. The assay developed herein has been designed, established and demonstrated to be suitable for the screening of α-synuclein aggregation inhibitors. The assay quantitatively measures aggregation using α- synuclein site-specifically labelled with green and red fluorescent dyes. Proteins labelled with the green dye are bound to microbeads. α-synuclein labelled with the red dye aggregates on the bead-linked green α-synuclein. The first part of the thesis describes the development of the tools required for the assay. α-synuclein single cysteine mutants were produced to introduce a specific attachment point to the protein. Single isomer carboxytetramethylrhodamine was synthesised in large scale for the label. Two different trifunctional tags that allow both the fluorescent labelling of the protein and the addition of a group for bead attachment in a single step were synthesised. Optimisation of the attachment of the functionalised proteins to beads of differing materials was accomplished enabling further development of the bead-based aggregation assay. With all tools established, the second part of the work comprised the development of the bead-based α-synuclein aggregation assay. Solid supports made of two different materials, TentaGel and Agarose, with two different types of bead surface attachment chemistry for α-synuclein were investigated, Ni-NTA on bead with His6-tag on the target or dibenzylcyclooctyne on bead and azide conjugation for the target. Only the combination of Ni-NTA agarose beads linking to His6-tag functionalised α-synuclein was found to be suitable for quantitative measurement of the aggregation process. Using 20 % EtOH, α-synuclein on-bead aggregation was reproducible within a 5 h time-frame with a linear dependence of aggregation rate as function of protein concentration on-bead. The third part of the thesis describes the research into novel starting points for the discovery of inhibitors of α-synuclein aggregation. In the peptides field, the most active peptides in the literature were selected and synthesised for study under the same conditions to find the most active ones. The most active peptide could be modified with non-natural amino acids to increase affinity and stability. While peptides and peptidomimetics would be applied in mechanistic studies, small molecular inhibitors of aggregation might represent lead compounds. One known inhibitor of α-synuclein aggregation was selected, NPT200-5, and an on-bead synthesis was developed so a diversity library could be generated around its four different building blocks. Finally the peptides, the NPT200-5 amide derivative and some known small molecule inhibitors of α-synuclein aggregation, such as curcumin, baicalein and EGCG amongst others, were screened on the bead-based α-synuclein aggregation assay. Strong inhibitory effects of curcumin and baicalein demonstrated the efficacy of the newly developed assay. In summary, the tools for the development of a novel micro-bead-based α-synuclein aggregation assay have been successfully produced. A novel bead-based α-synuclein early stage aggregation assay has been developed and optimised. Validation of this new technique was achieved with known small molecules inhibitors of α-synuclein aggregation.

Made available in DSpace on 2014-07-29T15:29:10Z (GMT). No. of bitstreams: 1 Dissert_EbersonSRN.pdf: 1230800 bytes, checksum: 70200ae7fcfb893a1baed80412086408 (MD5) Previous issue date: 2011-04-29 / Dementia is a neurodegenerative syndrome by impressive and also by the high occurrence rates of morbidity in caregivers. Dementia with Lewy bodies is a complex neuropsychiatric frame member, considered the second cause of degenerative dementia in the elderly, and resulting impact on quality of life in caregivers. The objectives of this study were to identify in the literature, the impact of caregiving on the quality of life for caregivers of a patient with dementia to describe the strategies used by health professionals and researchers in Brazil or to mitigate the negative impacts of caring for the patient dementia and assessing quality of life for families living with and caring for patients with dementia with Lewy bodies. It was initially performed an integrative review the electronic databases of the National Library of Medicine (PubMed) and Latin American Health Sciences (LILACS), the interval from 1999 to 2010. Then a cross-sectional study evaluated 90 caregivers of patients with dementia with Lewy bodies, responding to a sociodemographic questionnaire and the instrument for assessing the quality of life, WHOQOL-BREF. The integrative review included 19 articles, showing that despite high rates of stress, anxiety and physical and mental burden of home caregivers of patients with dementia, there is little national scientific output in this regard. Strategies were listed health professionals in two papers, with proof of the applicability and effectiveness of these interventions help the needs of caregivers. The field study showed that caregivers were mostly men, with two primary school education, married, average age 47.4 years ± 13.8 years and average time as a caregiver of 13.9 ± 9.3 months. The highest average scores for quality of life was in the Physical Domain and the lowest in the psychological and Environment. Variables associated with quality of life were age and duration of caregiver. Caregivers aged 60-75 years had the lowest scores in the Physical Domain. Those who care for less time had the greatest impact in the psychological environment and quality of life. We conclude that it is necessary to support the emotional aspects of the professional caregiver, allowing for better management of the situation. Knowledge and understanding of the specific symptoms of the disease helps the adequacy of personal resources to deal with behavioral changes, identified as the factor most impacting on the caregiver's life. Special attention should be given to changes in the caregiver's health, so it does not become a "hidden patient" and unable to cope with the demands of patients with dementia with Lewy bodies. / A demência é uma síndrome neurodegenerativa impactante pela grande ocorrência e também pelos índices de morbidade nos cuidadores. A demência com corpos de Lewy é um quadro neuropsiquiátrico complexo associado, considerado como segunda causa de demência degenerativa em idosos, e que provoca impacto na qualidade de vida nos cuidadores. Os objetivos deste estudo foram identificar na literatura, o impacto do cuidado na qualidade de vida de quem cuida de um portador de demência; descrever as estratégias utilizadas por profissionais de saúde e ou pesquisadores brasileiros para amenizar os impactos negativos decorrentes do ato de cuidar do portador de demência e avaliar a qualidade de vida de familiares que convivem e cuidam de portadores de demência com corpos de Lewy. Inicialmente foi realizada uma revisão integrativa nas bases eletrônicas da National Library of Medicine (PubMed) e Literatura Latino Americana de Ciências da Saúde (LILACS), no intervalo de 1999 a 2010. Em seguida foi realizado estudo transversal que avaliou 90 cuidadores de pacientes com demência com corpos de Lewy, respondendo a um questionário sociodemográfico e ao instrumento de avaliação da qualidade de vida, WHOQOL-BREF. A revisão integrativa incluiu 19 artigos, evidenciando que, apesar de altos os índices de estresse, ansiedade e desgaste físico e mental dos cuidadores domiciliares de pacientes com demência, é escassa a produção cientifica nacional a esse respeito. Foram apontadas estratégias dos profissionais de saúde em dois artigos, com aplicabilidade e comprovação da eficácia dessas intervenções no auxilio às necessidades dos cuidadores. O perfilsociodemográfico do estudo de campo evidenciou que os cuidadores entrevistados eram na maioria homens, com 2º grau de escolaridade, casados, de idade média 47,4 ± 13,8 anos anos e média de tempo como cuidador de 13,9 ± 9,3 meses. A maior média dos domínios de qualidade de vida foi no Domínio Físico e a menor nos Domínios Psicológico e Meio Ambiente. As variáveis associadas à qualidade de vida foram a idade e o tempo de cuidador. Os cuidadores com idade entre 60 - 75 anos apresentaram piores escores no Domínio Físico. Aqueles que cuidam há menos tempo tiveram maior impacto nos Domínios Psicológico e Meio Ambiente da qualidade de vida. Conclui-se que é necessário suporte profissional aos aspectos emocionais do cuidador, permitindo melhor gerenciamento da situação. O conhecimento das especificidades e compreensão dos sintomas da patologia auxiliam na adequação dos recursos pessoais para enfrentar as alterações comportamentais, apontadas como o fator mais impactante na vida do cuidador. Atenção especial deve ser dada às alterações na saúde do cuidador, para que ele não se torne um paciente oculto e incapaz de lidar com as demandas do portador de demência com corpos de Lewy.

Background: Lewy body dementia (LBD), consisting of Parkinson's disease dementia (PDD) and dementia with Lewy bodies (DLB), is known to make up more than 15% of dementia cases at autopsy, however the clinical prevalence rate is reported to be much lower at around 5-6%. Difficulties with diagnosis and/or lack of specific treatments may contribute to this difference. This study investigated the diagnosis and management pathways of LBD and whether inflammation could play a role in the pathophysiology and hence provide a route for future diagnostic and treatment pathways. Methods: Clinical diagnostic rates of LBD in clinics across several NHS trusts in East Anglia were reviewed, followed by an in-depth notes review of patients identified with LBD together with age and gender matched controls. A literature review of the current evidence for inflammation in LBD, preceded a case control study to investigate further. Nineteen DLB patients together with 16 age and gender matched healthy controls underwent [11C]PK11195 PET imaging, and the same cohorts, plus an additional 10 matched control subjects underwent peripheral cytokine analysis. Results: The clinical prevalence rate of LBD was low compared to the known pathology rates, with delays identified in the diagnosis of DLB compared to other dementia subtypes. Delays were also seen between the onset of dementia symptoms and the clinical diagnosis of dementia in Parkinson's disease (PD). The literature review identified studies providing evidence of inflammation in PD but few studies had been carried out in DLB. PET imaging revealed microglial activation negatively correlated with disease severity in DLB, suggesting inflammation occurs early in the disease. DLB patients also showed evidence of differences in cytokine levels compared to healthy controls. Conclusion: The study showed evidence of inflammatory changes in DLB, providing a potential target for treatment and/or biomarkers, that could assist in increasing clinical diagnostic rates.

Ce travail de thèse avait pour objectif principal de contribuer à une meilleure compréhension de la mémoire procédurale dans le vieillissement normal et pathologique. Plus spécifiquement, nous avons tenté de déterminer de manière plus précise limpact du mode dentraînement afin didentifier les conditions qui optimisent lacquisition de nouvelles habiletés dans le vieillissement normal et pathologique. Avant dexaminer plus précisément les implications de nos résultats sur les plans théorique et clinique, il convient tout dabord den faire un bref récapitulatif. LEtude 1 comportait un double objectif : (1) développer une épreuve évaluant lacquisition de nouvelles habiletés perceptivo-motrices qui serait facilement applicable dans un contexte clinique ; (2) étudier limpact du mode dentraînement sur lacquisition de ces nouvelles habiletés chez des sujets adultes. Dans cette perspective, nous avons créé une version informatisée du paradigme de dessin en miroir. Dans cette tâche, les sujets devaient parcourir des figures géométriques, le plus rapidement possible, à laide dune souris dordinateur inversée. Afin de poursuivre nos objectifs, trois expérimentations ont été menées. Dans lexpérience 1, deux types dentraînement étaient comparés : un entraînement constant et un entraînement variable. Nous pensions que ce dernier engendrerait de plus faibles performances durant la phase dacquisition, mais de meilleures performances lors de la phase test. Cependant, aucune différence de performance nest apparue entre les deux conditions. Nous avons, dès lors, supposé que cette absence de différence puisse être liée à la nature de linstruction donnée aux participants exigeant une grande rapidité. Ainsi, les sujets de la condition constante, parce quils réalisaient la tâche à la limite de leurs possibilités, se trouvaient de facto dans une condition comparable (en termes dinvestissement) à la condition variable. Dans lexpérience 2, une limitation de vitesse a été imposée sur les mêmes tâches afin de renforcer le contraste entre les conditions constante et variable. Cependant, aucune différence nest apparue entre les groupes. A nouveau, nous avons postulé que la méthodologie employée nétait pas en mesure de mettre en évidence une différence entre les modes dentraînement. En effet, durant lexpérimentation, il est apparu que les sujets des deux groupes commettaient de nombreuses erreurs engeandrant à leur tour un ralentissement du déplacement de la cible, obligeant donc les sujets à freiner et constamment contrôler leur mouvement. Par conséquent, il est possible que ce contrôle ait pu à nouveau rendre la condition constante comparable à la condition variable en termes dinvestissement cognitif. Nous avons dés lors proposé une nouvelle consigne durant la phase dacquisition de lexpérience 3 : réaliser le parcours en commettant le moins derreurs possible et sans contrainte de temps. Cette nouvelle adaptation na pas, une fois encore, permis de mettre en évidence de différence dapprentissage entre les diverses conditions dentraînement (constante, bloc et variable). Ces résultats suggèrent que la tâche de dessin en miroir informatisée était trop complexe pour mettre en avant un effet du mode dentraînement. LEtude 2 comportait également un double objectif : (1) créer une épreuve de lecture en miroir permettant dévaluer de manière plus précise lacquisition de nouvelles habiletés perceptivo-verbales ; (2) explorer plus spécifiquement ces capacités dans le vieillissement normal et dans la maladie dAlzheimer. En effet, après avoir remis en question la pertinence du paradigme de lecture en miroir de Cohen et Squire (1980), Masson (1986) a créé un nouveau paradigme mais navait, de façon étonnante, pas observé dapprentissage procédural chez des sujets jeunes. Dans ce contexte, nous avons apporté plusieurs modifications (lettres majuscules, non-mots, sens de lecture) à ce paradigme afin de le rendre plus efficace dans lévaluation de lapprentissage procédural. Ainsi, notre première expérience montre chez des sujets jeunes un apprentissage avec une amélioration des performances entre la phase dacquisition et la phase test tant pour des non-mots composés de lettres déjà traitées que pour des non-mots composés de nouvelles lettres encore jamais vues. Ces résultats suggéraient donc que la nouvelle version de cette tâche permettait dévaluer lapprentissage procédural et non plus un effet damorçage de répétition sur les lettres. Lors dune seconde expérience, nous avons donc mis à profit cette tâche pour évaluer lapprentissage de nouvelles habiletés perceptivo-verbales de manière plus fine et adaptée dans la maladie dAlzheimer. Les résultats ont mis en évidence un effet damorçage de répétition chez les patients et les sujets âgés de contrôle mais un apprentissage procédural uniquement chez les sujets âgés de contrôle. Cette incapacité pourrait remettre en question lidée générale selon laquelle lapprentissage procédural est généralement préservé dans cette pathologie. Toutefois, la difficulté des patients à acquérir cette habileté pourrait être liée à une trop grande variabilité de la tâche utilisée. Dès lors, administrer cette même épreuve selon un mode plus constant pourrait permettre aux patients, par une diminution du recours aux processus contrôlés, dacquérir cette habileté de lecture en miroir. Toujours dans le but dexplorer les capacités de mémoire procédurale dans le vieillissement pathologique, lEtude 3 a investigué cet aspect dans la démence à corps de Lewy (DCL). Bien quun intérêt grandissant est né pour cette pathologie depuis quelques années, aucune étude na jusquici tenté dy investiguer le statut de la mémoire procédurale. Les résultats ainsi obtenus montraient que les patients DCL, tout comme les sujets âgés, amélioraient leurs performances entre la phase dacquisition et la phase test tant pour les lettres déjà rencontrées que pour les nouvelles lettres. Dans lEtude 4, nous avons voulu déterminer si un mode dentraînement caractérisé par une plus faible variabilité, permettrait aux patients Alzheimer dacquérir lhabileté de lecture en miroir. Concrètement, cette étude comportait également un double objectif : (1) investiguer limpact du mode dentraînement (bloc versus variable) dans un domaine perceptivo-verbal chez des patients Alzheimer ; (2) tester lhypothèse selon laquelle un entraînement variable est caractérisé par un recours plus important au fonctionnement exécutif quun entraînement en bloc. Vingt-quatre patients Alzheimer ont été testés et deux conditions dentraînement ont été comparées. La condition en bloc consistait à présenter des non-mots construits avec une première série de lettres (série A) et ensuite présenter des non-mots avec une autre série de lettres (série B). En revanche, dans la pratique variable, lensemble des non-mots étaient construits avec des lettres appartenant aux séries A et B simultanément. Il est apparu que les patients issus de la condition en bloc lisaient les non-mots composés de nouvelles lettres significativement plus vite que le groupe variable, qui lui, na pas amélioré sa performance. De plus, nos résultats ont confirmé limplication du fonctionnement exécutif dans la pratique variable et non dans la pratique en bloc. Ces résultats montrent que des patients déficitaires sur le plan exécutif auront plus de difficultés à apprendre de nouvelles habiletés perceptivo-verbales au travers dun entraînement variable.

Les situations cliniques associant des troubles moteurs et une détérioration cognitive sont fréquentes et déroutantes pour le clinicien, qui est confronté à la difficile question du diagnostic différentiel entre plusieurs cadres nosographiques dont la maladie de Parkinson avec démence (MPD) et la démence à corps de Lewy Diffus (DCLD). Dans ce travail, nous avons étudié les caractéristiques neuropsychologiques pouvant différencier les deux affections. Nous avons, dans une première étude, montré que la mémoire de reconnaissance visuelle était altérée de façon différente. Dans un second travail, nous avons spécifié les caractéristiques de ces altérations en les rapprochant des classiques profils « cortical » et « sous cortical » de démence. Nous avons ainsi montré que la MPD présentait une altération de la mémoire de reconnaissance visuelle intermédiaire entre la Maladie d’Alzheimer et la DCLD. Enfin, dans une dernière partie, nous proposons des perspectives de recherche dans la continuité de ces travaux. / Clinical manifestations associating motor and cognitive impairment are frequently encountered and difficult for the clinician who is required to address the problem of making the correct differential diagnosis, particularly to differentiate Parkinson's disease with dementia (PDD) from Lewy bodies dementia (LBD). In this study, we examined the neuropsychological characteristics which allow us to differentiate the two disorders. In the first study, we demonstrated that visual recognition memory is disturbed differently in the two cases. In a second study, we specified the characterisstics of the modifications encountered by using the classic "cortical" and "subcortical" dementia profiles. We also showed that, in PDD, the alteration in visual recognition memory is intermediary between Alzheimer's disease and LBD. Finally, in the last part of our study, we suggest future avenues of research needed to complete our work.

Les synucléinopathies regroupent les maladies neurodégénératives de Parkinson, les démences à corps de Lewy et l'atrophie multi-systématisée. Des études suggèrent que les synucléinopathies seraient des maladies à prion. Aujourd'hui, certains aspects manquent pour que l'α-synucléine soit reconnue comme un prion. Par exemple, il est à démontrer que chaque synucléinopathie est causée par une souche précise d'α-synucléine. Durant ma thèse j’ai mis au point une méthode d'amplification fiable des dépôts présents dans le cerveau des patients atteints de synucléinopathies. J’ai aussi documenté les procédures de nettoyage à adopter envers des matériels souillés, par diverses fibres amyloïdes, afin de réduire le risque de contamination. Finalement, j’ai été associé à une étude montrant les capacités de propagation d'assemblages d'α synucléine, dans un réseau de neurones humains en culture. Ces résultats permettront des études structurales, et fonctionnelles, des souches d’α-synucléine dans les synucléinopathies. / The aggregation of α-synuclein protein has been shown to be associated with Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, called synucleinopathies. Increasing amount of evidences suggest that synucleinopathies are prion diseases. Some aspects are missing for α-synuclein to be recognized as a prion, such as the existence of strains associated to synucleinopathies. During my thesis I set up a reliable method to amplify α-synuclein-rich deposits from patients tissues. I validated the method using all synucleinopathies tissues. This should allow the identification of α-synuclein strain related to each synucleinopathy. In addition, I also documented cleaning procedures for materials soiled with various amyloid fibers, in order to reduce the risk of contamination. Finally, I was associated to a study that shows the propagation abilities of different α-synuclein assemblies in a neuronal network mimicking human cortico-cortical connections. These results open the way to structural and functional studies of the amplified deposits.

The protein alpha-synuclein (α-synuclein) accumulates in the brain in disorders such as Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). It is believed that the monomeric form of α-synuclein can adopt a partially folded structure and start to aggregate and form intermediately sized oligomers or protofibrils. The aggregation process can continue with the formation of insoluble fibrils, which are deposited as Lewy bodies. The oligomers/protofibrils have been shown to be toxic to neurons and are therefore believed to be involved in the pathogenesis of the actual diseases.       The overall aims of this thesis were to investigate the properties of α-synuclein oligomers and to generate and characterize antibodies against these species. In addition, the potential for immunotherapy of the α-synuclein oligomer-selective antibodies were evaluated in a transgenic mouse model with α-synuclein pathology. Stable, β-sheet rich α-synuclein oligomers were induced by incubation with either one of the reactive aldehydes 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE). The oligomers exhibited distinct morphological properties, although both types were toxic when added to a neuroblastoma cell line. The seeding effects of ONE-induced oligomers were studied in vitro and in vivo. The oligomers induced seeding of monomeric α-synuclein in a fibrillization assay but not in a cell model or when injected intracerebrally in transgenic mice. It seemed, however, as if the oligomers affected α-synuclein turnover in the cell model. By immunizing mice with HNE-induced oligomers antibody producing hybridomas were generated. Three monoclonal antibodies were found to have strong selectivity for α-synuclein oligomers. These antibodies recognized Lewy body pathology in brains from patients with PD and DLB as well as inclusions in the brain from young α-synuclein transgenic mice, but did not bind to other amyloidogenic proteins. Finally, immunotherapy with one of the oligomer/protofibril selective antibodies resulted in lower levels of such α-synuclein species in the spinal cord of α-synuclein transgenic mice. To conclude, this thesis has focused on characterizing properties of α-synuclein oligomers. In particular, antibodies selectively targeting such neurotoxic forms were generated and evaluated for passive immunization in a transgenic mouse model. Such immunotherapy may represent a future treatment strategy against Lewy body disorders.

No description available.