La bioingénierie de greffons endothéliaux cornéens est une des solutions réalistes en cours de développement dans quelques laboratoires pour palier au grand déséquilibre entre pénurie mondiale de dons de cornée et besoins immenses et croissant des populations. Cette véritable médecine régénérative consistera à injecter des cellules endothéliales (CEs) dans la chambre antérieure aux stades précoces des dystrophies endothéliales et, pour les stades avancées des pathologies endothéliales, à reconstruire in vitro des greffons endothéliaux composés d'un support transparent et biocompatible colonisé par une monocouche des CEs. Dans les 2 cas, la première étape obligatoire est la production de masse de CEs ou de "CE-like". Dans ce travail, nous avons exploré les différentes possibilités pour obtenir suffisamment de CEs fonctionnelles pour envisager des applications cliniques : 1- L'expansion de CEs natives prélevées sur des cornées de donneurs, ou culture primaire, se heurtent aux capacités prolifératives limitées des CEs in vitro. Un des prérequis étant la compréhension des mécanismes d'arrêt de la prolifération des CEs humaines, nous avons fait la synthèse bibliographique des connaissances sur leur cycle cellulaire et leur sénescence, et présentons 2 articles originaux: le premier utilise un microarray spécifique de 112 gènes de contrôle du cycle cellulaire pour comparer les profils transcriptionnels des CEs de 6 modèles biologiques comportant théoriquement un stimulus prolifératif croissant: in vivo, post mortem, organoculture, culture primaire confluente, culture primaire non confluente et lignée immortalisée. Nous identifions de nombreux acteurs impliqués dans l'arrêt du cycle, en particulier ceux impliquant une réponse à des dommages oxydatifs de l'ADN. Le second article explore les capacités prolifératives résiduelles des CEs de donneurs âgés de plus de 50 ans, sont les plus nombreux en Europe et donc les pourvoyeurs habituels de CEs pour la bioingénierie. Nous montrons qu'une optimisation des techniques de culture permet d'obtenir in vitro une mosaïque endothéliale de 2000 cellules/mm2. Enfin, un troisième article montre qu'un stimulus physique inattendu constitué d'un train d'impulsion électrique permet d'obtenir un très grand nombre de figures mitotiques mais uniquement dans des zones de faible DCE, démontrant encore une fois l'importance majeur de l'inhibition de contact dans l'arrêt prolifératif. 2- La sélection et l'expansion, par culture en sphère, des progéniteurs endothéliaux de la périphérie endothéliale pourrait être un moyen de contourner la sénescence de la majorité des CEs. La synthèse bibliographique montre que la plupart des travaux publiés émanent d'une seule équipe japonaise. Dans notre second article, nous avons montré l'existence de rares "label-retaining cells" dans les cornées de donneurs âgés, qui pourraient correspondre à ces progénieteurs. Nous avons aussi montré qu'il était possible d'obtenir des sphères avec ces donneurs. 3- La différenciation de cellules souches embryonnaires, mésenchymateuses ou des cellules pluripotentes induites est enfin la troisième voie qui pourrait permettre de produire des CEs en grand nombre. La méthode d'induction de la différenciation pourrait reproduire le schéma physiologique et désormais bien caractérisé de formation de l'endothélium à partir du mésenchyme périoculaire dérivé de la crête neurale et que nous rappelons au début de notre mémoire bibliographique. Nous rappelons également les méthodes utilisables pour caractériser les cellules obtenues: vérification de leur identité par immunomarquage d'un panel de protéines caractéristique (en absence de marqueur unique spécifique) et vérification de leur fonctionnalité par mesures de leurs capacités de pompage ionique, au minimum de façon indirecte sur chambre d'électrophysiologie de type Ussing, au mieux de façon directe en quantifiant la déturgescence d'une cornée humaine conservée dans le bioréacteur breveté du BiiGC / Corneal endothelial engineering is becoming a more and more realistic solution to restore vision from corneal edema. This method focus to regenerate corneal endothelium by direct injection of corneal endothelial cells (ECs) into patient anterior chamber at the early stage of endothelial dystrophies, or by grafting a transparent biocompatible material covered by a monolayer of ECs. These two techniques require both in vitro isolation and amplification of ECs or endothelial-like cells. In this thesis, different strategies to obtain a high quantity of functional ECs for clinical application are explored: 1- Due to the limit proliferative capacity of EC, the first strategy consists to analyze mechanisms implicated EC cell cycle arrest and then to optimize protocol for native EC isolation or for cell proliferation activation ex vivo. This is summarized in three publications. The first publication describes the cell cycle regulation by comparing transcriptional expression of 112 genes in 6 biological models of EC with different proliferative profile: in vivo, postmortem, organ-culture, confluent primary culture, non confluent primary culture and immortalized cell line. , The key molecular actors identified using the combining microarray analysis and gene ontology methods are consistent with previous findings about oxidative DNA damage mechanism. The second publication characterizes EC differentiation process and its impact on EC proliferative capacity in old donor corneas. Analyses of differentiation/progenitor markers and of proliferative capacity underline the differentiation process of EC from the centre to the peripheral corneal endothelium. Thereby, an optimized culture protocol was developed, allowing the formation of high-density monolayer (> 2000 cells/mm2) with stable endothelial morphology. We proved the possibility to make profit from a majority of old-donor cornea grafts invalidated for penetrating graft In the third publication, the activation of endothelial cell cycle by electric pulses directly in corneal graft was characterized. We confirm the activation of endothelial cell cycle at different phases but also the damage of tissue during electroporation. 2- Second strategy consists of the amplification of ECs from potential EC progenitors. Using sphere forming culture and a new method to detect slow-cycling cells, we demonstrate the existence of "young" ECs population with higher proliferative capacity in corneal periphery. The isolation of ECs by sphere formation is one possible step for ECs selection in vitro. 3- The differentiation of embryonic stem cells, mesenchymal stem cells or induced pluripotent stem cells into corneal endothelial cells is the third approach considered by our laboratory. The manipulation of stem cells differentiation would be based on the molecular mechanisms implicated in the formation of corneal endothelium from periocular mesenchymal cells described in the first part of the bibliography. Finally, in order to validate the quality of endothelial cell mass obtained, we revisited recent methods for the evaluation of corneal endothelial identity (immunolocalisation of specific markers), for the measurement of pump activity of cell monolayer (Ussing chamber, perfusion chamber) or directly in deswelled cornea using the bioreactor patented by the BiiGC laboratory

In cultured medaka testis fragments, cells remained viable for the entire culture period (17h), and spermatids that developed from spermatocytes were viable and motile. Primary cultures were characterized over a period of two days with respect to cell viability and the distribution of adherent and suspended cells. These two cell populations were maintained in dynamic equilibrium in vitro for several days. Proliferating cells were predominant among clusters of suspended cells, as determined by BrdU labeling, and CFSE and propidium iodide PI labeling. Based on cytological criteria, the proliferating cells were mostly spermatogonia and possibly also preleptotene spermatocytes. Differentiation of spermatocytes into spermatids or spermatozoa was also observed, mainly among the suspended cells. These results suggest that the organ and primary culture systems are suitable systems for studying the effects of substances that interfere with spermatogenesis in the medaka, a model vertebrate. The organ and primary culture systems were used to analyze the effects of a synthetic estrogen, EE2, on cell proliferation in medaka testis. Both organ and primary culture were suitable for this purpose consistently small concentrations (0.01 and 1 nM) of EE2 stimulated cell proliferation slightly, while higher concentrations (100 nM) had an inhibitory effect. To investigate the effect of phytoestrogens on cell proliferation in spermatogenesis, selected flavonoids [genistein (1, 10, 100 µg/ml), quercetin (0.01, 1, 100 µM), and 8-prenylnarigenin (0.001, 0.1, 1, 10 µM)] were added to medaka testis primary cultures. Genistein and quercetin inhibited cell proliferation in the cultures while 8-prenylnarigenin had no effect. In a second series of experiments the addition of genistein (10 µg/ml) to primary cultures significantly inhibited both cell proliferation and cell differentiation as determined by flow cytometry using CFSE/PI labeling.

Os mecanismos etiopatogênicos que levam ao desenvolvimento dos nódulos e tumores da tireoide ainda não são bem conhecidos. É fato estabelecido que a prevalência dessas lesões é maior nas mulheres. Dessa forma, o que nos motivou a realizar esta Tese foi avaliar se os hormônios sexuais femininos tem efeito direto sobre as células de tireoide humanas normais podendo contribuir para a sua etiopatogenia. Este estudo teve como objetivos padronizar um modelo de cultura primária de células foliculares de tireoide humana normal e nesse modelo validar um gene normalizador após tratamento com estradiol e progesterona, avaliar os efeitos da progesterona na expressão dos genes NIS, TG e TPO bem como avaliar a expressão gênica e proteíca e a possível localização intracelular do receptor de membrana GPR30 nestas células. Em nosso modelo de cultura primária em monocamada, as células foliculares mantiveram sua morfologia cubóide característica e permaneceram diferenciadas o que foi evidenciado pela dosagem de tireoglobulina e T4 livre, no sobrenadante do meio de cultura, e pela identificação das proteínas TG e TPO por imunocitoquímica. A estimulação com progesterona aumentou a expressão dos genes NIS, TG e TPO, respectivamente, 1.78 (p=0.003), 1.50 (p=0.034) e 1.64 (p=0.018) vezes, quando comparadas ao grupo tratado somente com TSH. Essa estimulação da progesterona foi inibida por mifepristona sugerindo que a progesterona tem efeito direto nas células foliculares da tireoide e que esse efeito é mediado por seu receptor nuclear. A normalização da expressão gênica foi realizada pelo gene β-actina, o qual demonstrou uma maior estabilidade entre os grupos analisados. Demonstramos também que as células normais da tireoide expressam tanto o gene como a proteína do receptor de membrana GPR30 com possível localização na membrana celular e no espaço perinuclear. / The mechanisms leading to the development of thyroid nodules and tumors are not well established. As these lesions are more common in women, female sex hormones could be involved in the pathogenesis of these disorders. The objectives of this study were to establish a model of primary culture of normal human thyroid follicular cells, to validate a normalizing gene for qRT-PCR after treatment with estradiol and progesterone, to evaluate the effects of progesterone on the expression of genes NIS, TG and TPO, and to evaluate the GPR30 gene and protein expression as well as its possible intracellular location, in these cells. In our model of primary monolayer culture, follicular cells maintained their characteristic cuboid differentiated morphology; and had evidence of differentiated thyroid function: the production of thyroglobulin and free T4, and identification of TG and TPO proteins by immunocytochemistry . Adding progesterone to TSH increased NIS, TG and TPO mRNA, respectively, 1.78 (p=0.003), 1.50 (p=0.034) and 1.64 (p=0.018) folds, compared to the group treated with only TSH. This stimulation was inhibited by mifepristone, suggesting that progesterone has a direct effect on the thyroid follicular cells. Normalization of gene expression was performed using β-actin as reference gene. We have also demonstrated that normal thyroid cells expressed GPR30 gene and protein, which is possibly localized in the plasma membrane and the perinuclear region.

Os mecanismos etiopatogênicos que levam ao desenvolvimento dos nódulos e tumores da tireoide ainda não são bem conhecidos. É fato estabelecido que a prevalência dessas lesões é maior nas mulheres. Dessa forma, o que nos motivou a realizar esta Tese foi avaliar se os hormônios sexuais femininos tem efeito direto sobre as células de tireoide humanas normais podendo contribuir para a sua etiopatogenia. Este estudo teve como objetivos padronizar um modelo de cultura primária de células foliculares de tireoide humana normal e nesse modelo validar um gene normalizador após tratamento com estradiol e progesterona, avaliar os efeitos da progesterona na expressão dos genes NIS, TG e TPO bem como avaliar a expressão gênica e proteíca e a possível localização intracelular do receptor de membrana GPR30 nestas células. Em nosso modelo de cultura primária em monocamada, as células foliculares mantiveram sua morfologia cubóide característica e permaneceram diferenciadas o que foi evidenciado pela dosagem de tireoglobulina e T4 livre, no sobrenadante do meio de cultura, e pela identificação das proteínas TG e TPO por imunocitoquímica. A estimulação com progesterona aumentou a expressão dos genes NIS, TG e TPO, respectivamente, 1.78 (p=0.003), 1.50 (p=0.034) e 1.64 (p=0.018) vezes, quando comparadas ao grupo tratado somente com TSH. Essa estimulação da progesterona foi inibida por mifepristona sugerindo que a progesterona tem efeito direto nas células foliculares da tireoide e que esse efeito é mediado por seu receptor nuclear. A normalização da expressão gênica foi realizada pelo gene β-actina, o qual demonstrou uma maior estabilidade entre os grupos analisados. Demonstramos também que as células normais da tireoide expressam tanto o gene como a proteína do receptor de membrana GPR30 com possível localização na membrana celular e no espaço perinuclear. / The mechanisms leading to the development of thyroid nodules and tumors are not well established. As these lesions are more common in women, female sex hormones could be involved in the pathogenesis of these disorders. The objectives of this study were to establish a model of primary culture of normal human thyroid follicular cells, to validate a normalizing gene for qRT-PCR after treatment with estradiol and progesterone, to evaluate the effects of progesterone on the expression of genes NIS, TG and TPO, and to evaluate the GPR30 gene and protein expression as well as its possible intracellular location, in these cells. In our model of primary monolayer culture, follicular cells maintained their characteristic cuboid differentiated morphology; and had evidence of differentiated thyroid function: the production of thyroglobulin and free T4, and identification of TG and TPO proteins by immunocytochemistry . Adding progesterone to TSH increased NIS, TG and TPO mRNA, respectively, 1.78 (p=0.003), 1.50 (p=0.034) and 1.64 (p=0.018) folds, compared to the group treated with only TSH. This stimulation was inhibited by mifepristone, suggesting that progesterone has a direct effect on the thyroid follicular cells. Normalization of gene expression was performed using β-actin as reference gene. We have also demonstrated that normal thyroid cells expressed GPR30 gene and protein, which is possibly localized in the plasma membrane and the perinuclear region.

Os mecanismos etiopatogênicos que levam ao desenvolvimento dos nódulos e tumores da tireoide ainda não são bem conhecidos. É fato estabelecido que a prevalência dessas lesões é maior nas mulheres. Dessa forma, o que nos motivou a realizar esta Tese foi avaliar se os hormônios sexuais femininos tem efeito direto sobre as células de tireoide humanas normais podendo contribuir para a sua etiopatogenia. Este estudo teve como objetivos padronizar um modelo de cultura primária de células foliculares de tireoide humana normal e nesse modelo validar um gene normalizador após tratamento com estradiol e progesterona, avaliar os efeitos da progesterona na expressão dos genes NIS, TG e TPO bem como avaliar a expressão gênica e proteíca e a possível localização intracelular do receptor de membrana GPR30 nestas células. Em nosso modelo de cultura primária em monocamada, as células foliculares mantiveram sua morfologia cubóide característica e permaneceram diferenciadas o que foi evidenciado pela dosagem de tireoglobulina e T4 livre, no sobrenadante do meio de cultura, e pela identificação das proteínas TG e TPO por imunocitoquímica. A estimulação com progesterona aumentou a expressão dos genes NIS, TG e TPO, respectivamente, 1.78 (p=0.003), 1.50 (p=0.034) e 1.64 (p=0.018) vezes, quando comparadas ao grupo tratado somente com TSH. Essa estimulação da progesterona foi inibida por mifepristona sugerindo que a progesterona tem efeito direto nas células foliculares da tireoide e que esse efeito é mediado por seu receptor nuclear. A normalização da expressão gênica foi realizada pelo gene β-actina, o qual demonstrou uma maior estabilidade entre os grupos analisados. Demonstramos também que as células normais da tireoide expressam tanto o gene como a proteína do receptor de membrana GPR30 com possível localização na membrana celular e no espaço perinuclear. / The mechanisms leading to the development of thyroid nodules and tumors are not well established. As these lesions are more common in women, female sex hormones could be involved in the pathogenesis of these disorders. The objectives of this study were to establish a model of primary culture of normal human thyroid follicular cells, to validate a normalizing gene for qRT-PCR after treatment with estradiol and progesterone, to evaluate the effects of progesterone on the expression of genes NIS, TG and TPO, and to evaluate the GPR30 gene and protein expression as well as its possible intracellular location, in these cells. In our model of primary monolayer culture, follicular cells maintained their characteristic cuboid differentiated morphology; and had evidence of differentiated thyroid function: the production of thyroglobulin and free T4, and identification of TG and TPO proteins by immunocytochemistry . Adding progesterone to TSH increased NIS, TG and TPO mRNA, respectively, 1.78 (p=0.003), 1.50 (p=0.034) and 1.64 (p=0.018) folds, compared to the group treated with only TSH. This stimulation was inhibited by mifepristone, suggesting that progesterone has a direct effect on the thyroid follicular cells. Normalization of gene expression was performed using β-actin as reference gene. We have also demonstrated that normal thyroid cells expressed GPR30 gene and protein, which is possibly localized in the plasma membrane and the perinuclear region.

In cultured medaka testis fragments, cells remained viable for the entire culture period (17h), and spermatids that developed from spermatocytes were viable and motile. Primary cultures were characterized over a period of two days with respect to cell viability and the distribution of adherent and suspended cells. These two cell populations were maintained in dynamic equilibrium in vitro for several days. Proliferating cells were predominant among clusters of suspended cells, as determined by BrdU labeling, and CFSE and propidium iodide PI labeling. Based on cytological criteria, the proliferating cells were mostly spermatogonia and possibly also preleptotene spermatocytes. Differentiation of spermatocytes into spermatids or spermatozoa was also observed, mainly among the suspended cells. These results suggest that the organ and primary culture systems are suitable systems for studying the effects of substances that interfere with spermatogenesis in the medaka, a model vertebrate. The organ and primary culture systems were used to analyze the effects of a synthetic estrogen, EE2, on cell proliferation in medaka testis. Both organ and primary culture were suitable for this purpose consistently small concentrations (0.01 and 1 nM) of EE2 stimulated cell proliferation slightly, while higher concentrations (100 nM) had an inhibitory effect. To investigate the effect of phytoestrogens on cell proliferation in spermatogenesis, selected flavonoids [genistein (1, 10, 100 µg/ml), quercetin (0.01, 1, 100 µM), and 8-prenylnarigenin (0.001, 0.1, 1, 10 µM)] were added to medaka testis primary cultures. Genistein and quercetin inhibited cell proliferation in the cultures while 8-prenylnarigenin had no effect. In a second series of experiments the addition of genistein (10 µg/ml) to primary cultures significantly inhibited both cell proliferation and cell differentiation as determined by flow cytometry using CFSE/PI labeling.

Este trabalho investigou, pela primeira vez, a influência do CH na modulação do metabolismo e proliferação de fibroblastos dérmicos humanos (FDHs) derivados de áreas fotoprotegida e fotoexposta, cultivados em modelo de monocamada. Além disto, foram investigados os efeitos da suplementação com CH na secreção de colágeno tipo I, em modelo de cultura 3D de equivalente dérmico, derivado de matriz produzida exclusivamente por FDHs. O tratamento com CH não influenciou a proliferação celular dos fibroblastos derivados de ambas as áreas, porém modulou expressivamente o metabolismo dos FDHs cultivados em monocamada, elevando o conteúdo de pró-colágeno I e colágeno I e diminuindo a atividade de metaloproteinases de matriz (MMP) 1 e 2. Concentrações menores de CH foram suficientes para estimular as células de área fotoexposta, sugerindo efeitos mais pronunciados do CH nestas células. Este estudo é uma contribuição importante para compreensão dos efeitos biológicos do CH nas células da pele e viabilidade do seu uso como ingrediente funcional de suplementos alimentares. / This study investigated, for the first time, the influence of CH on the extracellular matrix metabolism and proliferation of human dermal fibroblasts (HDFs) derived from sun-protected and sun-exposed body sites, cultured in monolayer in vitro model. Moreover, CH effects on the secretion of type I collagen were investigated in dermal equivalent 3D model derived from dermal matrix produced exclusively by HDFs. CH treatment did not affect cellular proliferation of either cell cultures, but notably modulated cell metabolism in monolayer model, increasing the content of procollagen I and collagen I and decreasing metalloproteinase activity (MMP) 1 and 2. These effects were confirmed in the human dermal equivalent model. Lower concentrations of CH were enough to stimulate sun-exposed-derived HDFs, suggesting more pronounced effect in these cells. This study presents an important contribution to understanding the biological effects of CH in skin cells and viability of its use as a functional ingredient in food supplements.

No description available.

Este trabalho investigou, pela primeira vez, a influência do CH na modulação do metabolismo e proliferação de fibroblastos dérmicos humanos (FDHs) derivados de áreas fotoprotegida e fotoexposta, cultivados em modelo de monocamada. Além disto, foram investigados os efeitos da suplementação com CH na secreção de colágeno tipo I, em modelo de cultura 3D de equivalente dérmico, derivado de matriz produzida exclusivamente por FDHs. O tratamento com CH não influenciou a proliferação celular dos fibroblastos derivados de ambas as áreas, porém modulou expressivamente o metabolismo dos FDHs cultivados em monocamada, elevando o conteúdo de pró-colágeno I e colágeno I e diminuindo a atividade de metaloproteinases de matriz (MMP) 1 e 2. Concentrações menores de CH foram suficientes para estimular as células de área fotoexposta, sugerindo efeitos mais pronunciados do CH nestas células. Este estudo é uma contribuição importante para compreensão dos efeitos biológicos do CH nas células da pele e viabilidade do seu uso como ingrediente funcional de suplementos alimentares. / This study investigated, for the first time, the influence of CH on the extracellular matrix metabolism and proliferation of human dermal fibroblasts (HDFs) derived from sun-protected and sun-exposed body sites, cultured in monolayer in vitro model. Moreover, CH effects on the secretion of type I collagen were investigated in dermal equivalent 3D model derived from dermal matrix produced exclusively by HDFs. CH treatment did not affect cellular proliferation of either cell cultures, but notably modulated cell metabolism in monolayer model, increasing the content of procollagen I and collagen I and decreasing metalloproteinase activity (MMP) 1 and 2. These effects were confirmed in the human dermal equivalent model. Lower concentrations of CH were enough to stimulate sun-exposed-derived HDFs, suggesting more pronounced effect in these cells. This study presents an important contribution to understanding the biological effects of CH in skin cells and viability of its use as a functional ingredient in food supplements.

Les maladies neurodégénératives liées à l’âge, telle celle de Parkinson, sont un problème majeur de santé publique. Cependant, la maladie de Parkinson reste incurable et les traitements sont très limités. En effet, les causes de la maladie restent encore mal comprises et la recherche se concentre sur ses mécanismes moléculaires. Dans cette étude, nous nous sommes intéressés à deux phénomènes anormaux se produisant dans la maladie de Parkinson : l’agrégation de l’α-synucléine et l’activation des cellules microgliales. Pour étudier la polymérisation de l’α-synucléine, nous avons établi de nouvelles méthodes permettant la production in vitro de différents types d’oligomères d’α-synucléine. Grâce à des méthodes biophysiques de pointe, nous avons caractérisé ces différents oligomères à l’échelle moléculaire. Puis nous avons étudié leurs effets toxiques sur les neurones. Ensuite, nous nous sommes intéressés à l’activation des microglies et en particulier à leurs canaux potassiques et aux changements liés au vieillissement. Nous avons identifié les canaux Kv1.3 et Kir2.1 et montré qu’ils étaient impliqués dans l’activation des microglies. En parallèle, nous avons établi une méthode originale qui permet l’isolation et la culture de microglies primaires issues de cerveaux adultes. En comparaison à celles de nouveaux-nés, les microglies adultes montrent des différences subtiles mais cruciales qui soutiennent l’hypothèse de changements liés au vieillissement. Globalement, nos résultats suggèrent qu’il est possible de développer de nouvelles approches thérapeutiques contre la maladie de Parkinson en modulant l’action des microglies ou en bloquant l’oligomérisation de l’ α-synucléine. / Age-related neurodegenerative disorders like Parkinson’s disease take an enormous toll on individuals and on society. Despite extensive efforts, Parkinson’s disease remains incurable and only very limited treatments exist. Indeed, Parkinson’s pathogenesis is still not clear and research on its molecular mechanisms is ongoing. In this study, we focused our interest on two abnormal events occurring in Parkinson’s patients, namely α-synuclein aggregation and microglial activation. We first investigated α-synuclein and its abnormal polymerisation. For this purpose, we developed novel methods, which allowed the in vitro production of different types of α-synuclein oligomers. Using highly sensitive biophysical methods, we characterised these different oligomers at a single-particle level. Then, we tested their biological effects on neurons. Afterwards, we studied microglial activation. We concentrated our efforts on two axes, namely age-related changes in microglial function and K+ channels in microglia. We showed that Kv1.3 and Kir2.1 K+ channels are involved in microglial activation. In parallel, we developed a new approach, which allows the effective isolation and culture of primary microglia from adult mouse brains. Adult primary microglia presented subtle but crucial differences in comparison to microglia from neo-natal mice, confirming the hypothesis of age-related changes of microglia. Taken together, our results support the hypotheses that microglial modulation or inhibition of α-synuclein oligomerisation are possible therapeutic strategies against Parkinson's disease.