Les donneurs de méthyles (B12 et folates) régulent le cycle des monocarbones qui joue un rôle primordial dans les régulations épigénétiques/épigénomiques par méthylation. Une carence en donneurs de méthyles produit un retard de croissance intra-utérine et favorise les anomalies du développement, principalement du système nerveux central. De plus, des taux élevés d'homocystéine associés à une telle carence constituent un facteur de risque pour diverses pathologies neurodégénératives. Nous avons étudié les conséquences d'une carence péri-conceptionnelle et gestationnelle sur le développement cérébral embryonnaire de rats Wistar. L'étude morphométrique a montré un retard de croissance des embryons carencés qui affectait également le cerveau, avec une atrophie de structures telles que l'hippocampe, le cortex et la zone subventriculaire. En raison de la forte sensibilité de l'hippocampe, les effets de la carence ont par ailleurs été étudiés sur un modèle cellulaire de progéniteurs neuronaux hippocampiques. L'utilisation de ces deux modèles a permis de montrer in vivo et in vitro la régulation négative par la carence de la voie Stat3, qui influence prolifération et survie, via une régulation épigénomique post-transcriptionnelle impliquant miR-124. La dérégulation du programme neurogénique impliquant les histones désacétylases affecte la différenciation cellulaire. Par ailleurs, nous avons démontré que la carence en donneurs de méthyles était associée à une modification post-traductionnelle correspondant à une N-homocystéinylation irreversible de protéines neuronales, en particulier associées au cytosquelette. Cette modification induit l'agrégation des protéines, phénomène impliqué dans de nombreuses maladies neurodégénératives. La combinaison de ces différents mécanismes apporte un éclairage nouveau sur les défauts de développement et les troubles cognitifs associés à une carence précoce en donneurs de méthyles, soulignant l'importance de la « programmation foetale » dans la survenue de certaines pathologies neurologiques / Methyl donors (B12 and folate) regulate the one-carbon cycle that plays an important role in the epigenetic/epigenomic regulations by methylation. Methyl donor deficiency (MDD) leads to intrauterine growth retardation and promotes neurodevelopmental abnormalities. Also, high levels of homocysteine associated with such a deficiency are a risk factor for various neurodegenerative diseases. We have studied the consequences of a periconceptional and gestational deficiency on the development of the embryonic brain of Wistar rats. Morphometric studies showed retardation in the development of deficient embryos which also affected the brain, with an atrophy of some structures including hippocampus, cortex and subventricular zone. Given the high sensitivity of the hippocampus, the effects of MDD have been additionally studied in a cellular model of hippocampal neuronal progenitors. Using these two models, we showed both in vivo and in vitro the downregulation of Stat3 pathway regulating cell proliferation and survival, through an epigenomic post-transcriptional process involving miR-124. Disruption of the neurogenic program implying histone deacetylases was shown to alter cell differentiation. Furthermore, we showed that methyl donor deficiency was associated with a post-translational modification corresponding to an irreversible N- homocysteinylation of neuronal proteins, especially those associated with the cytoskeleton. Such a process leads to protein aggregation, a phenomenon involved in many neurodegenerative diseases. The combination of these different mechanisms provides new insights into developmental defects and cognitive impairment associated with an early MDD, highlighting the importance of "fetal programming" in the occurrence of some neurological diseases

No description available.

Les neurodégénérescences avec accumulation de fer dans le cerveau (Neurodegeneration with Brain Iron Accumulation, NBIA) sont des maladies neurodégénératives progressives, génétiquement hétérogènes. On connait actuellement 11 gènes de ces maladies mais pour la plupart d’entre eux leur lien avec l’accumulation en fer est encore incompris. Ce travail de thèse présente deux nouveaux gènes de NBIA identifiés par séquençage d’exome dans deux familles indépendantes. Le premier gène, REPS1, est impliqué dans le recyclage de l’endosome. Les fibroblastes de patients sont caractérisés par une accumulation de fer qui est corrigée par l’expression de l’ADNc de REPS1 dans ces cellules. Le deuxième gène, CRAT, code une carnitine acétyltransferase et le déficit de β-oxydation détecté dans les fibroblastes du patient a été corrigé par l’expression de l’ADNc CRAT normal. Le rôle de REPS1 dans le recyclage de l’endosome a mis sur la voie du mécanisme physiopathologique des NBIA. En effet, les fibroblastes des patients REPS1 et CRAT mais aussi d’autres patients avec des mutations d’autres gènes connus de NBIA (PANK2, PLA2G6, FA2H, C19ORF12) ont une accumulation massive en fer et une anomalie de recyclage du récepteur à la transferrine (TfR1). TfR1 permet l’entrée du fer par endocytose et son expression est régulée par le contenu en fer des cellules. La seule régulation connue de l’homéostasie du fer se fait au niveau post-transcriptionnel par le système IRP/IRE qui est fonctionnel dans les fibroblastes NBIA alors que la protéine TfR1 s’accumule. Cette accumulation de fer montre ainsi qu’il existe une régulation post-traductionnelle, jusqu’ici inconnue, et qui n’est pas fonctionnelle dans les NBIA. Nous avons pu montrer que cette régulation se faisait par une palmitoylation du TfR1, déficitaire dans les NBIA, mais restaurée par l’artesunate. Ainsi quel que soit le gène muté, tous les NBIA résultent d’une anomalie de recyclage du TfR1 permettant de les définir comme des maladies du trafic intracellulaire. La deuxième partie de la thèse s’intéresse au mécanisme physiopathologique de l’ataxie de Friedreich (FRDA) caractérisée elle aussi par une accumulation de fer dans le cerveau. FRDA est due à des expansions de triplets dans le premier intron du gène FXN conduisant à l’extinction de FXN et de PIP5K1B situé en amont. L’étude de modèles cellulaires dans lesquels le gène FXN et/ou PIP5K1B ont été éteints par siRNA et de fibroblastes de patients a permis de mettre en évidence une anomalie de l’homéostasie du fer qui rappelle celle observée dans les NBIA. L’ensemble de ces résultats a permis de comprendre le mécanisme physiopathologique des NBIA, de mettre à jour une régulation encore inconnue de l’homéostasie du fer mais aussi d’envisager une voie de traitement des NBIA. / Neurodegeneration with brain iron accumulation (NBIA) encompasses a group of rare neurogenerative disorders with different clinical and molecular features, underlined by progressive extrapyramidal dysfunction and iron accumulation in the brain. To date, mutations in 11 genes are currently known. Nevertheless for most of them their link with iron accumulation is still misunderstood. This work presents two novel NBIA genes identified by exome sequencing in two independent families. The first gene, REPS1, is involved in endosome recycling. Patient’s fibroblasts are characterized by iron overload corrected by wild-type REPS1 cDNA overexpression. The second gene, CRAT, encodes a carnitine acetyltransferase and a β-oxidation deficit in patient’s fibroblasts has been fixed by overexpression of wild-type CRAT cDNA. The function of REPS1 in endosome recycling put on the path of the NBIA pathophysiological mechanism. Indeed, fibroblasts of REPS1 patients but also from other patients mutated in various NBIA genes (CRAT, PANK2, PLA2G6, FA2H, C19ORF12) present massive iron accumulation and abnormal transferrin receptor (TfR1) recycling. TfR1 allows iron uptake by endocytosis and its expression is regulated by the iron cellular status. The only known regulation of iron homeostasis occurs at the posttranscriptional level by the IRE/IRP system which is functional in NBIA fibroblasts whereas TfR1 protein accumulates. This iron accumulation highlights a yet unknown posttranslational regulation which is not functional in NBIA. We have been able to demonstrate that this regulation occurs via TfR1 palmitoylation, which is defective in NBIA, but restored by artesunate. Hence, whatever the disease gene, all NBIA gave rise to abnormal TfR1 recycling which allows defining NBIA as intracellular trafficking disease. The second part of the thesis focused on the pathophysiological mechanism of the Friedreich ataxia (FRDA) also characterized by brain iron overload . FRDA is related to triplets expansions in the first intron of FXN gene leading to the extinction of FXN and PIP5K1B upstream gene. Studying cellular models knocked down for FXN and/or PIP5K1B by siRNA and patients’ fibroblasts of patients allowed to detect abnormal iron homeostasis reminiscent of NBIA. All these results allowed to decipher the NBIA pathophysiological mechanism, to highlight a yet unknown iron homeostasis regulation and to open possible ways towards therapeutic drugs for NBIA.

No description available.

As doenças neurodegenerativas (DN) estão entre as principais causas de mortalidade e morbidade nos países ocidentais. Não há ainda um tratamento definitivo para estas neuropatias, mas estudos têm indicado mecanismos comuns de toxicidade que incluem disfunção mitocondrial, estresse oxidativo, apoptose e neuroinflamação. Adicionalmente, o efeito benéfico da neuroplasticidade induzida por fatores neurotróficos no retardamento ou inibição do processo neurodegenerativo também tem sido sugerido por muitos estudos. O beta-cariofileno é um sesquiterpeno bi-cíclico encontrado no óleo essencial de algumas plantas, e que possui efeito anti-inflamatório e antioxidante. Assim, este composto possui características e é capaz de induzir efeitos que o tornam um potencial candidato ao tratamento/prevenção dos processos envolvidos na neurodegeneração. Apesar disso, pouco se sabe sobre os efeitos e os mecanismos de ação do beta-cariofileno no processo de degeneração neuronal. Então, neste estudo, avaliou-se o efeito do beta-cariofileno em modelos celulares (PC 12) de neurotoxicidade que mimetizam in vitro os mecanismos moleculares envolvidos nas doenças de Parkinson, Huntington e Alzheimer, os quais, para efeitos práticos, denominaremos de \"modelos celulares de Parkinson, Huntington e Alzheimer\". Estes modelos são induzidos experimentalmente pela neurotoxina dopaminérgica iodeto de 1-metil 4-fenil piridina (MPP+), pela neurotoxina mitocondrial ácido 3-nitropropiônico (3NP) e pelo peptídeo neurotóxico B-amiloide (AB42), respectivamente. O beta-cariofileno apresentou efeitos benéficos nestes três modelos de neurotoxicidade, e adicionalmente induziu neuritogênese e a expressão de proteínas neurotípicas no modelo neuronal. Este é o primeiro estudo a demonstrar tais efeitos do beta-cariofileno. / Neurodegenerative diseases (ND) are among the leading causes of mortality and morbidity in Western countries. There is not a definitive treatment for these neuropathies, but studies have indicated common mechanisms of toxicity that include mitochondrial dysfunction, oxidative stress, neuroinflammation and apoptosis. Additionally, the beneficial effect of the neuroplasticity induced by neurotrophic factors on the retardation or inhibition of neurodegeneration has also been suggested by several studies. Beta-caryophyllene is a bicyclic sesquiterpene found in essential oils of some plants, and possesses anti-inflammatory and antioxidant effects. Thus, this compound has characteristics and is capable of inducing effects that make it a potential candidate for treatment / prevention of the processes involved in neurodegeneration. Despite this, little is known about the effects and mechanisms of action of beta-caryophyllene in the neuronal degeneration process. Then, this study evaluated the effect of beta-caryophyllene in cellular models of neurotoxicity (PC 12) that mimic in vitro the molecular mechanisms involved in Parkinson\'s, Huntington\'s and Alzheimer\'s diseases, which, for practical purposes, we will denominate \"Cellular models of Parkinson\'s, Huntington\'s and Alzheimer\'s diseases.\" These models are experimentally induced by the dopaminergic neurotoxin 1-methyl iodide, 4-phenyl pyridine (MPP+), by the mitochondrial neurotoxin 3-nitropropionic acid (3NP) and the neurotoxic peptide B-amyloid (AB42), respectively. Beta-caryophyllene showed beneficial effects on these three models of neurotoxicity, and additionally induced neuritogenesis and the expression of neurotypic proteins in the neuronal model. This is the first study to demonstrate such effects of beta-caryophyllene.

Septinas constituem uma família de proteínas de ligação a GTP que foram inicialmente identificadas em levedura Saccharomyces cerevisiae, mas também estão presentes em outros eucariotos com exceção de plantas. Septinas são purificadas de leveduras, Drosophila e cérebros de mamíferos na forma de filamentos, porém o mecanismo através do qual acorre a formação destes filamentos ainda não é muito bem compreendido. Septinas são constituídas de três regiões principais: um N-terminal variável, um domínio central GTPase altamente conservado e um domínio coiled-coil C-terminal. O gene SEPT4 foi identificado por M. Tanaka e colaboradores a partir do cDNA de cérebro humano e apresentou duas distintas transcrições: Bradeiona ? e ?. Interessantemente, além de cérebro e coração, as proteínas Bradeiona Α e Β. são detectadas somente em câncer do cólon, reto, próstata e melanoma maligno. Neste trabalho, o gene da proteína Bradeiona Β foi subclonado em um vetor de expressão bacteriano, produzido em E. coli e purificado com sucesso. O espectro de dicroísmo circular (CD) mostrou o perfil característico de proteínas com hélices a na estrutura secundária. Resultados de cromatografia de exclusão molecular (SEC) e espalhamento dinâmico de luz (DLS) indicam que a septina Bradeina foi produzida na forma de um estável oligômero com características monodispersivas, que foi subseqüentemente cristalizado em PEG6000. A atividade GTPase da Bradeiona Β foi comprovada através da técnica de eletroforese capilar (CE), mostrando-se absolutamente dependente de íons Mg2+. Inibição da atividade GTPase foi verificada em altas concentrações de Mg2+ (maiores que 5 mM). Com a finalidade de caracterizar os domínios preditos da Bradeiona Β (Fragmento Conservado e domínio GTPase), essas regiões foram previamente definidas, expressas em E. cozi e purificadas com sucesso. Resultados de CD, SEC, espectroscopia de fluorescência e NMR-600MHz indicam que o FC foi produzido na forma de um estável monômero com pouca estrutura secundária regular. Resultados de DLS e CD indicam que a fusão 6xHis-DGTPase foi produzida na forma de um oligômero com a presença de hélices a na estrutura secundária. A fusão 6xHis-DGTPase mostrou-se instável a altas concentrações na ausência de imidazol. A atividade GTPase da fusão GST+DGTPase foi comprovada, similarmente a Bradeiona , através da técnica de CE. Novamente, verificou-se dependência de íons Mg2+ (para a atividade catalítica) e inibição em altas concentrações de Mg2+. A fusão GST+DGTPase também foi capaz de hidrolisar ATP. Espera-se que as informações relatadas neste estudo proporcionem um alicerce para estudos estruturais/funcionais futuros das proteínas Bradeiona Α e Βoutras septinas. / Septins form a class of eukaryoyic guanine nucleotide-binding proteins that were first identified in budding yeast. Septins purified from yeast, Drosophila and mammalian brain form filaments, however the mechanism by which the filaments assemble is unclear. Septins have a highly conserved structure, which includes a central GTP-binding domain, a variable N-terminal region, and most septins also contain a coiled coil domain at the Cterminus. Bradeion p is one of the splice variants of the human septin gene, SEPT4, recently isolated by expression screening of an adult human brain cDNA library. The Bradeion gene resides at 17q23, and has been shown to present specific expression in both human colorectal cancer, urologic cancers and malignant melanoma. In order to characterize the GTPase activity of Bradeion Β , the protein was successfully expressed in E. coli and purified. The recombinant protein was characterized by circular dichroism (CD), dynamic light scattering (DLS) and a novel non-radioactive enzyme assay, which utilizes capillary electrophoresis (EC) to monitor GTP hydrolysis. The CD spectrum exhibited the typical shape characteristic of the presence of helical elements of secondary structure and the DLS pattern was indicative of a monodisperse solution, which was readily crystallized in the presence of PEG6000. GTP hydrolysis was shown to be Mg2+ dependent within the low millimolar range but at 5 mM was inhibitory. In order to characterize the predicted domains of Bradeion Β, these defined regions were successfully expressed in E. cozi and purified. The CD spectrum of CF exhibited the shape typically found for non-regular structure. The results of fluorescence spectroscopy, gel filtration (SEC) and NMR-600MHz also corroborate with the CD results indicating an irregular structure. The fusion protein 6xHis-DGTPase exhibited a CD spectrum with the typical shape characteristic of the presence of helical elements but was show to be instable at high concentrations in the absence of imidazole. To characterize the GTPase activity of the fusion protein GST+DGTPase, the CE technique was used to monitor GTP hydrolysis. Analysis by CE showed that GST+DGTPase was functional, since both GTP and ATP hydrolysis was observed in a Mg2+ dependent manner. This work provides novel approaches, which should aid in the fbture study of the structure and fùnction of Bradeion Α e Β, others septins and related GTPases.

A neurodegeneração é um processo onde ocorre morte celular progressiva. O tráfego neuronal anterógrado e retrógado, e entre os compartimentos é essencial para a viabilidade celular. As proteínas Rabs pertencem à família de pequenas GTPases, com funções de tráfego de vesículas e organelas, para realizarem sua função as proteínas Rab podem recrutar proteínas motoras como as KIF 1B e KIF 5, responsáveis pelo transporte anterógrado mitocondrial. A associação do distúrbio do tráfego intracelular com doenças neurodegenerativas tem sido tema de estudos recentes. Com isso o objetivo do presente trabalho é analisar a expressão das proteínas Rab, bem como estudar as proteínas motoras que podem contribuir para o esclarecimento sobre os distúrbios no tráfego intracelular que antecedem a formação de agregados proteicos envolvidos em neurodegeneração. Para tanto, utilizou-se o modelo de tratamento com rotenona para indução de agregados em Ratos Lewis idosos que foram expostos a rotenona durante 4 semanas, em seguida foram avaliados os níveis de expressão das proteínas Rab no hipocampo, substância negra e locus coeruleus, por western blotting. Foram analisados também os níveis de expressão das proteínas motoras KIF1B e KIF5 antes e durante a formação de agregados proteicos, em culturas de células, de ratos Lewis neonatos, do hipocampo, substância negra e locus coeruleus tratadas com rotenona por 24 horas ou 48 horas nas concentrações de 0,1nM, 0, 3nM e 0,5nM. Foi observado diminuição dos níveis de expressão das proteínas Rab 1 nas regiões do hipocampo e locus coeruleus. Houve aumento de expressão das Rab 4,5 e 6 no hipocampo, porém na substância negra a expressão da Rab 1 aumentou e da Rab 6 diminuiu. Já no locus coeruleus in vivo a Rab 6 aumentou, mas as Rab 1, 5 e 11 diminuíram sua expressão. Já a expressão da KIF 5 aumentou com o tratamento de 0,1nM de rotenona e diminuiu após 0,5nM do xenobiótico por 48 horas in vitro, na mesma região. Na substância negra aumentaram as KIFs 1B e 5 após o tratamento com 0,5nM por 48 horas in vitro, mas diminuíram as KIF 1B e 5 após o tratamento com 0,3nM por 24 horas e KIF 5 após o tratamento com 0,1nM por 48 horas. Esses resultados permitem concluir que a expressão de proteínas importantes para o tráfego mitocondrial e de vesículas encontram-se alteradas e fazem parte dos eventos intracelulares que antecedem a neurodegeneração / Neurodegeneration is a process that leads to progressive cell death. The anterograde and retrograde neuronal traffic as well as the traffic between compartments are essential for cell viability. The Rab proteins belong to the small GTPases family with function of vesicles and organelle trafficking. Rab proteins can recruit motor proteins such as KIF 1B and KIF 5 that are responsible for anterograde mitochondrial transport. The association of intracellular traffic disturb with neurodegenerative diseases have been theme of recent studies. Thereat the objective of this study is analyze the expression of Rab and motor proteins that can contribute for the understanding about the disturb of the intracellular traffic that precedes protein aggregation involved in neurodegeneration. For this purpose it was employed the model of rotenone treatment for induction of aggregation in aged Lewis rats that were exposed to rotenone during 4 weeks in order to evaluate Rabs expression. The levels of motor proteins KIF 1B and KIF 5 expression were evaluated before and during the formation of protein aggregates in hippocampus, substantia nigra and locus coeruleus cell cultures of neonates Lewis rats, exposed to rotenone for 24 hours or 48 hours in the concentrations of 0.1nM, 0.3nM or 0.5nM. It was observed decreased levels of Rab 1 expression in hippocampus and locus coeruleus. Rabs 4,5 and 6 were increased in the hippocampus, but in the substantia nigra the expression of Rab 1 increased and Rab 6 decreased. In the locus coeruleus the Rab 6 increased, but Rabs 1, 5 and 11 decreased. The expression of KIF 5 increased after 0.1nM of rotenone and decreased after the exposure to 0.5nM of for 48 hours in cultured cell from the locus coeruleus. In the substantia nigra the KIF1B and KIF 5 increased after treatment with 0.5nM for 48 hours in vitro, but these protein decreased after treatment with 0.3nM for 24 hours in vitro, and KIF 5 after treatment with 0.1nM for 48 hours. These results allow us conclude that the expression of important proteins for the mitochondrial and vesicles traffic are altered and participate of intracellular events that precede the neurodegeneration

Wong, Siu Lun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 129-141). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract (Chinese version) --- p.iii / Acknowledgements --- p.iv / List of Abbreviations --- p.v / List of Tables --- p.vii / List of Figures --- p.viii / Chapter 1. --- INTRODUCTION / Chapter 1.1 --- Neurodegenerative disorders - a brief overview --- p.1 / Chapter 1.2 --- Polyglutamine diseases --- p.2 / Chapter 1.3 --- Microscopically visible polyglutamine protein aggregates and its relation to toxicity --- p.7 / Chapter 1.4 --- Polyglutamine protein conformers and their relation to toxicity --- p.10 / Chapter 1.5 --- Modeling polyglutamine diseases in Drosophila / Chapter 1.5.1 --- GAL4/UAS spatial transgene expression system in Drosophila --- p.14 / Chapter 1.5.2 --- Temporal control of GAL4/UAS transgene expression system in Drosophila --- p.16 / Chapter 1.5.3 --- Drosophila as a model to study human pathologies --- p.19 / Chapter 1.5.4 --- Drosophila as a model to study polyglutamine diseases --- p.21 / Chapter 1.6 --- Aims of study --- p.26 / Chapter 2. --- MATERIALS AND METHODS / Chapter 2.1 --- Drosophila culture and manipulation / Chapter 2.1.1 --- Drosophila culture --- p.27 / Chapter 2.1.2 --- Phenotypic examination of adult external eye degeneration --- p.27 / Chapter 2.1.3 --- Pseudopupil assay of adult retinal degeneration and observation of green fluorescent protein in adult eyes --- p.28 / Chapter 2.2 --- Semi-quantitative Reverse Transcription-Polymerase Chain Reaction / Chapter 2.2.1 --- RNA extraction from adult Drosophila heads --- p.30 / Chapter 2.2.2 --- DNase treatment of extracted RNA --- p.31 / Chapter 2.2.3 --- Reverse transcription-Polymerase Chain Reaction (RT-PCR) --- p.31 / Chapter 2.2.4 --- Agarose gel electrophoresis --- p.33 / Chapter 2.3 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) / Chapter 2.3.1 --- Protein extraction from adult Drosophila heads --- p.33 / Chapter 2.3.2 --- Preparation of SDS-polyacrylamide gel and electrophoresis --- p.34 / Chapter 2.3.3 --- Western blotting --- p.35 / Chapter 2.3.4 --- Immunodetection --- p.36 / Chapter 2.4 --- Immunoprecipitation --- p.38 / Chapter 2.5 --- Filter retardation assay --- p.39 / Chapter 2.6 --- Isolation and solubilization of SDS-insoluble protein --- p.40 / Chapter 2.7 --- Sucrose gradient sedimentation --- p.41 / Chapter 2.8 --- Preparation of Drosophila tissues for immunofluorescence analysis / Chapter 2.8.1 --- Dissection and immunostaining of Drosophila larval imaginal eye discs --- p.42 / Chapter 2.8.2 --- Cryosectioning and immunostaining of adult Drosophila heads --- p.44 / Chapter 2.9 --- Atomic force microscopy --- p.47 / Chapter 2.10 --- Reagents and buffers / Chapter 2.10.1 --- Reagents for Drosophila culture --- p.48 / Chapter 2.10.2 --- Reagents for RT-PCR --- p.52 / Chapter 2.10.3 --- Reagents for SDS-PAGE --- p.54 / Chapter 2.10.4 --- Reagents for immunoprecipitation --- p.57 / Chapter 2.10.5 --- Reagents for filter retardation assay --- p.57 / Chapter 2.10.6 --- Reagents for isolation and solubilization of SDS-insoluble protein --- p.58 / Chapter 2.10.7 --- Reagents for sucrose gradient sedimentation --- p.58 / Chapter 2.10.8 --- Reagents for immunofluorescence --- p.59 / Chapter 3. --- RESULTS / Chapter 3.1 --- Establishment of an inducible transgenic Drosophila model of polyglutamine diseases / Chapter 3.1.1 --- Introduction --- p.60 / Chapter 3.1.2 --- Results / Chapter 3.1.2.1 --- GAL80ts-mediated inducible expression of expanded polyglutamine protein in Drosophila / Chapter 3.1.2.1.1 --- GAL80ts controls GAL4/UAS-mediated polyQ protein expression --- p.61 / Chapter 3.1.2.1.2 --- Inducible expression of SDS-soluble expanded polyglutamine protein --- p.64 / Chapter 3.1.2.1.3 --- Inducible expression of expanded polyglutamine protein accumulates gradually in form of SDS-insoluble protein --- p.66 / Chapter 3.1.2.1.4 --- Inducible expression of expanded polyglutamine protein results in progressive accumulation of microscopically visible aggregates --- p.68 / Chapter 3.1.2.2 --- Inducible expression of expanded polyglutamine protein causes late-onset progressive neuronal degeneration in Drosophila / Chapter 3.1.2.2.1 --- Inducible expression of expanded polyglutamine protein leads to late-onset progressive deterioration of photoreceptor neurons --- p.68 / Chapter 3.1.2.2.2 --- Inducible expression of expanded polyglutamine protein neither causes external eye degenerative phenotype nor disrupts gross retinal morphology despite deterioration of photoreceptor neurons --- p.72 / Chapter 3.1.2.3 --- Co-expression of caspase inhibitor P35 suppresses polyglutamine-induced neuronal degeneration --- p.72 / Chapter 3.1.2.4 --- Co-expression of molecular chaperone Hsp70 suppresses polyglutamine-induced neuronal degeneration --- p.74 / Chapter 3.1.2.5 --- Inducible expression of expanded polyglutamine protein results in biphasic expression of molecular chaperone Hsp70 in Drosophila --- p.76 / Chapter 3.1.3 --- Discussion --- p.76 / Chapter 3.2 --- Involvement of microscopically visible polyglutamine aggregates in neurodegeneration / Chapter 3.2.1 --- Introduction --- p.83 / Chapter 3.2.2 --- Results / Chapter 3.2.2.1 --- Effect of Hsc70-K71S on microscopically visible polyglutamine aggregates and neuronal degeneration / Chapter 3.2.2.1.1 --- Co-expression of Hsc70-K71S reduces the level of microscopically visible polyglutamine aggregates --- p.83 / Chapter 3.2.2.1.2 --- Co-expression of Hsc70-K71S does not alter polyglutamine transgene expression --- p.84 / Chapter 3.2.2.1.3 --- Co-expression of Hsc70-K71S does not modify polyglutamine-induced neuronal degeneration --- p.87 / Chapter 3.2.2.2 --- Microscopically visible polyglutamine aggregates do not correlate with neuronal degeneration --- p.90 / Chapter 3.2.3 --- Discussion --- p.93 / Chapter 3.3 --- Detection of small SDS-insoluble expanded polyglutamine protein species and its association with neurodegeneration / Chapter 3.3.1 --- Introduction --- p.97 / Chapter 3.3.2 --- Results / Chapter 3.3.2.1 --- Accumulation of SDS-soluble expanded polyglutamine protein does not correlate with neuronal degeneration --- p.98 / Chapter 3.3.2.2 --- Identification of small SDS-insoluble expanded polyglutamine protein species / Chapter 3.3.2.2.1 --- Accumulation of total SDS-insoluble expanded polyglutamine protein positively correlates with progressive neuronal degeneration --- p.99 / Chapter 3.3.2.2.2 --- Accumulation of large SDS-insoluble expanded polyglutamine protein does not correlate with neuronal degeneration --- p.99 / Chapter 3.3.2.2.3 --- Accumulation of small SDS-insoluble expanded polyglutamine protein correlates with neuronal degeneration --- p.104 / Chapter 3.3.3 --- Discussion --- p.107 / Chapter 3.4 --- Biophysical characterization of small SDS-insoluble expanded polyglutamine protein species / Chapter 3.4.1 --- Introduction --- p.109 / Chapter 3.4.2 --- Results / Chapter 3.4.2.1 --- Separation of expanded polyglutamine protein species by sucrose gradient sedimentation --- p.110 / Chapter 3.4.2.2 --- Morphological studies of small SDS-insoluble expanded polyglutamine protein species by atomic force microscopy --- p.112 / Chapter 3.4.3 --- Discussion --- p.118 / Chapter 4. --- GENERAL DISCUSSION --- p.124 / Chapter 5. --- CONCLUSION --- p.127 / Chapter 6. --- REFERENCES --- p.129

A confocal scanning laser ophthalmoscope (CSLO) was used to image the axonal and dendritic aborizations of RGCs in the Thy-1 YFP mice. With quantitative analysis of cell body area, axon diameter, dendritic field, number of terminal branches, total dendritic branch length, branching complexity, symmetry and distance from the optic disc, the morphologies of RGCs and the patterns of axonal and dendritic degeneration were analyzed. After optic nerve crush, RGC damage was observed prospectively to begin with progressive dendritic shrinkage, followed by loss of the axon and the cell body. Similar pattern of RGC degeneration was observed after 90 minutes of retinal ischemia although no morphological changes were detected when the duration of ischemia was shortened to 30 minutes. The rate of dendritic shrinkage was variable and estimated on average 2.0% per day and 11.7% per day with linear mixed modeling, after optic nerve crush and retinal ischemic injury, respectively. RGCs with a larger dendritic field had a slower rate of dendritic shrinkage. / In summary, we demonstrated that dendritic shrinkage could be evident even before axonal degeneration after optic nerve crush and retinal ischemic injury. We have established a methodology for in vivo and direct visualization of RGCs and retinal microglia, which could provide reliable and early markers for neuronal damage. Measuring the rate of dendritic shrinkage and tracking the longitudinal activation of microglia would provide new paradigms to study the mechanism of neurodegenerative diseases and offer new insights in testing novel therapies for neuroprotection. / Progressive neuronal cell death and microglial activation are the key pathological features in most neurodegenerative diseases. While investigating the longitudinal profiles of neuronal degeneration and microglial activation is pertinent to understanding disease mechanism and developing treatment, analyzing progressive changes has been obfuscated by the lack of a non-invasive approach that allows long term, serial monitoring of individual neuronal and microglial cells. Because of the clear optical media in the eye, direct visualization of the retinal ganglion cells (RGCs) and microglia is possible with high resolution in vivo imaging technique. In this study, we developed experimental models to visualize and characterize the cellular morphology of RGCs and retinal microglia in vivo in the Thy-1 YFP and the CX3CR1 +/GFP transgenic mice, described the patterns of axonal and dendritic shrinkage of RGCs, discerned the dynamic profile of microglial activation and investigated the relationship between RGC survival and microglial activation after optic nerve crush and retinal ischemic injury induced by acute elevation of intraocular pressure. / The longitudinal profile of microglial activation was investigated by imaging the CX3CR1GFP/+ transgenic mice with the CSLO. Activation of retinal microglia was characterized with an increase in cell number reaching a peak at a week after optic nerve crush and retinal ischemic injury, which was followed by a gradual decline falling near to the baseline at the 4 th week. The activation of retinal microglia was proportional to the severity of injury. The number of RGCs survival at 4 weeks post-injury was significantly associated with the number of activated retinal microglia. / Li, Zhiwei. / Adviser: Leung Kai Shun. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 50-66). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Les similitudes entre les mécanismes neurotoxiques responsables des encéphalopathies spongiformes Transmissibles (EST) et des synucléinoapthies, ainsi que la présence concomitante des formes pathologiques de la protéine prion et de l’α-synucléine au sein d’une même maladie neurodégénérative sont deux observations qui nous ont conduits à étudier les relations existant potentiellement entre les altérations moléculaires de l’α-synucléine et les maladies à prion. Après avoir développé des anticorps monoclonaux en immunisant avec de l’α-synucléine recombinante humaine des souris n’exprimant pas de façon endogène cet immunogène, nous avons caractérisé les altérations moléculaires de l’α-synucléine apparaissant conjointement à une symptomatologie motrice sévère lors du vieillissement de souris transgéniques (TgM83) surexprimant l’α-synucléine humaine mutée en A53T. Les essais d’inoculation intracérébrale de souris TgM83 par différentes souches de prion ont mis en évidence que la transmission de l’encéphalopathie spongiforme bovine de type H permet de déclencher chez ces animaux une maladie à prion de façon concomitante au développement d’altérations moléculaires de l’α-synucléine. Enfin, l’importante accélération de la pathologie liée a l’α-synucléine observée chez des souris TgM83 ayant été inoculées par des tissus contenant des formes altérées de l’α-synucléine, constitue un résultat soutenant le fait que la pathologie liée a l’α-synucléine serait capable de se propager expérimentalement de proche en proche, comme la protéine prion pathologique au cours des EST / The overlap of neurotoxic mecanisms involved in prion diseases and synucleinopathies, and the concomitant detection of pathological forms of prion and α-synuclein in a same neurodegenerative disease, raise questions about the existence of potential relationship between α‐synuclein molecular alteration and prion diseases. First, we developed monoclonal antibodies by immunizing mice presenting a spontaneous deletion of the α-synuclein gene with human recombinant α‐synuclein. Then, we characterized the molecular alterations appearing jointly to clinical signs during the aging of a transgenic mouse model of synucleinopathies (TgM83), overexpressing human A53T α‐synuclein. Then, an approach routinely done in the field of prion was used to trigger a synucleinopathy alongside a prion disease. For this purpose, TgM83 mice were inoculated intracerebrally by three different prion strains : transmission of H-type bovine spongiform encephalopathy allows the onset of a prion disease concomitantly to the α‐synuclein pathology developed by the TgM83 mouse model. Finally, intracerebral inoculation of TgM83 mice with brain homogenates from symptomatic mice affected by a synucleinopathy triggers an important acceleration of the α‐synuclein pathology, resulting in the early onset of motor clinical signs associated with molecular alterations of α-synuclein. These data suggest that α-synuclein alterations can be experimentally transmitted from one mouse to another, supporting the idea that, far from being confined to the transmissible spongiform encephalopathies, the « prion-like » propagation of misfolded neuronal proteins might occur in synucleinopathies