Role of Lmx1a and Lmx1b transcription factors in post-mitotic midbrain dopaminergic neurons

Lmx1a et Lmx1b sont des facteurs de transcription connus pour leur rôle au cours du développement des neurones dopaminergiques du mésencéphale (mDA). Ils ont été montrés comme essentiels à chacune des étapes de différentiation des progéniteurs en neurones dopaminergiques matures. Des études récentes ont également mis en évidence l'importance de ces deux facteurs de transcription dans les neurones dopaminergiques chez l'adulte. Lmx1a/b sont impliqués dans la régulation de gènes mitochondriaux ainsi que dans l'autophagie. Cependant, jusqu'à présent, rien n'est connu sur le rôle de Lmx1a/b dans les neurones dopaminergiques post-mitotiques. Le but de cette thèse est d'élucider le rôle de Lmx1a/b dans les neurones dopaminergiques matures. L'analyse des projections axonales dopaminergiques de souris doubles conditionnelles mutantes (cKO) pour Lmx1a/b a mis en évidence un défaut de guidage axonal confirmant le rôle essentiel de ces deux facteurs de transcription dans la formation des circuits dopaminergiques. Afin d’identifier précisément les molécules impliquées dans la régulation du système dopaminergique des techniques adaptées doivent être développées pour déterminer les principaux acteurs régulés par Lmx1a/b. À cette fin, nous avons mis au point une technique de marquage immunohistochimique rapide de la tyrosine hydroxylase (TH, enzyme nécessaire à la synthèse de la dopamine) sur des sections de mésencéphale de souris afin de délimiter la région d’intérêt. Par la suite, nous utilisons une technique de microdissection au laser afin de spécifiquement récolter les cellules dopaminergiques du mésencéphale pour réaliser un profil d'expression génique. Un premier article de méthodologie a été publié concernant cette technique. Cette procédure menée sur des souris cKO pour Lmx1a et Lmx1b et leurs contrôles associés a permis de mettre en évidence des gènes régulés par Lmx1a et Lmx1b tels que Plxnc1. Plxnc1 est une protéine de guidage axonal ayant pour ligand la sémaphorine 7a (Sema7a). Afin d'observer si la régulation de Plxnc1 par Lmx1a/b est à l'origine du défaut de guidage axonal observé chez les souris cKO pour Lmx1a/b, nous avons réalisé une analyse in vitro de l’effet de la Sema7a sur les axones d'explants mDA. Notre étude a montré un effet chimiorépulsif de la Sema7a pour les axones des neurones mDA exprimant Plxnc1. De plus, l’étude de souris Sema7a KO montre une augmentation de l’innervation DA dans la partie dorsale du striatum, partie exprimant Sema7a chez des souris contrôles. Ce phénotype met en évidence une chimiorépulsion induite par l’interaction Sema7a/Plxnc1. L’étude de souris surexprimant Plxnc1 a, quant à elle, montré une perte d’innervation DA dans la partie dorsale du striatum. En effet, la majorité des cellules du mésencéphale se mettent à exprimer Plxnc1, les rendant ainsi sensibles à la chimiorépulsion induite par Sema7a. L’ensemble de ces résultats met en évidence l’importance de la régulation de la protéine de guidage axonal Plxnc1 par Lmx1a/b pour l'innervation des cibles du mésencéphale. La répression de Plxnc1 dans les neurones dopaminergiques de la substance noire pars compacta (SNpc) semble nécessaire à l’innervation du striatum dorsal riche en Sema7a. Cette étude est la première à identifier les bases moléculaires du guidage axonal expliquant la ségrégation des voies mDA nigrostriée et mésolimbique, et devrait contribuer à améliorer l'efficacité des thérapies cellulaires pour la maladie de Parkinson. Un second article sera soumis prochainement sur le rôle des facteurs de transcription Lmx1a/b dans les neurones dopaminergiques post-mitotiques du mésencéphale. La principale caractéristique histopathologique de la maladie de Parkinson est la dégénérescence des neurones mDA de la SNpc. La thérapie de remplacement cellulaire utilisant des neurones dopaminergiques nouvellement générés à partir de cellules souches représente une thérapie prometteuse. Cependant, la mauvaise innervation des neurones nouvellement greffés limite le succès des études de transplantation. L’identification de facteurs régulant la connectivité des neurones mDA devient primordiale pour élucider les mécanismes impliqués dans la mise en place du système dopaminergique. C'est pourquoi, dans une derniere partie, afin d'illustrer cette possibilité d'amélioration d'une thérapie de remplacement cellulaire, j’ai réalisé l’implantation de cellules souches différenciées en neurones dopaminergiques dans un modèle de souris lésées à la 6-hydroxydopamine (6OHDA). Les cellules nouvellement réimplantées sont de type SNpc, en raison de l'infection par un vecteur viral induisant l'inhibition de l'expression de Plxnc1.Lmx1a and Lmx1b are transcription factors known for their role in the development of midbrain dopamine neurons (mDA). They were shown as essential for each stage of differentiation from progenitors to mature dopaminergic neurons. Recent studies have also highlighted the importance of these two transcription factors in dopaminergic neurons in adult mice. Lmx1a/b are involved in the regulation of mitochondrial genes and in autophagy. Although some evidence suggest that they could be involved in the formation of mDa circuit formation, their role in post-mitotic mDA neurons remains unknown. The aim of this thesis is to elucidate the role of Lmx1a/b in post-mitotic dopaminergic neurons. Analysis of dopaminergic axonal projections of double conditional mutant (cKO) mice for Lmx1a/b showed an axon guidance defect confirming the essential role of these transcription factors in the formation of dopaminergic circuits. In order to precisely identify the molecules involved in the regulation of the dopamine system, suitable techniques must be developed to identify the main genes that are regulated by Lmx1a/b. To this end we developed a new technique allowing gene profiling of brain sub-population. By combining rapid immunolabeling of mDA neurons with laser capture microdissection we manage to extract RNA from two sub-regions of mDA neurons such as ventral tegmental area (VTA) and substantia nigra pars compacta (SNpc). The advantage of this technique is to compare quickly the regulation of genes expression by studying controls and mutant mice. A first methodological article has been published regarding this procedure. We then applied this technique on cKO mice for Lmx1a/b and their associated controls to identify genes regulated by Lmx1a and Lmx1b. Among these genes, we identified Plxnc1, an axon guidance receptor for the semaphorin 7a (Sema7a). In order to verify whether the regulation of Plxnc1 by Lmx1a/b is at the origin of the axon guidance defect observed in double conditional mutant for Lmx1a/b, we have made an in vitro analysis of the effect of Sema7a on mDA explants. Our study showed a chemorepulsive effect of Sema7a on Plxnc1 positives axons. In addition, the study of knockout mice for Sema7a shows an increase of DA innervation in the dorsal part of the striatum which is the region expressing Sema7a in control mice. This phenotype reveals a chemorepulsion induced by Sema7a/Plxnc1 interaction. The study of mice overexpressing Plxnc1 shows a loss of DA innervation in the dorsal striatum. Indeed, by overexpressing Plxnc1, the majority of midbrain cells begin to express this axon guidance protein instead of only mDA neurons from the VTA. Thus, all mDA neurons including neurons from the SNpc express Plxnc1 making them sensitive to Sema7a. This interaction Sema7a/Plxnc1 leads to a chemorepulsion of axons guided away from the dorsal striatum. Overall these results highlight the importance of the regulation of the axon guidance protein Plxnc1 by Lmx1a/b for the innervation of midbrain targets. The repression of Plxnc1 expression in dopaminergic neurons of the SNpc appears necessary for the innervation of dopaminergic axons in the dorsal striatum, rich in Sema7a. This study is the first to identify the molecular basis of the development of the dopaminergic system explaining the segregation of the nigrostriatal and mesolimbic pathways. These results should help to improve the effectiveness of cell therapies for Parkinson's disease. A second article will be submitted soon about the role of Lmx1a/b transciption factors in post-mitotic midbrain dopaminergic neurons. The main histopathological feature of Parkinson's disease (PD) is the degeneration of SNpc neurons. The cell replacement therapy using newly generated dopaminergic neurons from stem cells represents a promising therapy. However, a poor innervation of the newly grafted neurons limits the success of transplantation studies. The identification of factors regulating neuronal connectivity of mDA neurons becomes essential to elucidate the mechanisms involved in the establishment of the dopaminergic system. Therefore, in a final section of this thesis, I report preliminary study about cell replacement therapy in PD mouse model. I differentiated DA neurons from stem cells, knock-down Plxnc1 expression and performed grafting in 6-hydroxydopamine (6OHDA) mouse model to illustrate the possibility of improving a cell replacement therapy

Relevance of lab-scale conical twin-screw extruder for thermoplastic STARCH/PLA blends rheology study

During the last decade, thermoplastic starch (TPS) has been studied more and more, alone and in blends. One key property of TPS is its viscosity. A lab-scale conical twin-screw extruder has been used to process polymers or blends of polymers at a small scale (7 cm3), and to measure their viscosity by pressure loss in a backflow channel. TPS has been tested at different temperatures (100-180°C) and for a 100-900s-1 shear rate range. One surprising result is the viscosity evolution of starch/glycerol blends (supposed to have become TPS before the rheology measurements) with temperature. An increase is observed between 120 and 140°C and then it decreases between 140 and 160°C. Therefore plasticization may happen only from 140°C. Blends of TPS with PLA and with different additives have also been studied. The additives are introduced in a second time, ten minutes after the TPS/PLA blend recirculation. Their influence on the blend viscosity is instantaneous. A viscosity increase after the additive introduction can be a clue for an improvement of the blend compatibilisation, as observed with CMC. Thanks to this micro-compounder, viscosity and behaviour of polymers can be evaluated easily even for TPS which needs shear to flow. Moreover, the solidified sample collected in the backflow channel at the end of the experiment can be used to test different properties like mechanical or thermal (DMTA, DSC) ones. Using this kind of lab-scale extruder represents then a good opportunity for preview screening studies

[Immunohistochemical characteristic of myoma tissue in patients with uterine leiomyoma after treatment with ulipristal acetate]

Background. Uterine leiomyoma is one of the most common benign tumors of the female genital organs. The main conservative treatment of leiomyoma is progesterone receptor blockers that suppress myoma growth and may lead to its regression. Objective. To study the immunohistochemical features of myoma tissue in patients with uterine leiomyoma after treatment with selective progesterone modulator - ulipristal acetate. Methods. Leiomyoma tissue obtained from 9 patients after ulipristal acetate treatment were investigated. Group for comparison - leiomyoma from patients without hormonal therapy. Immunohistochemical study of progesterone and estrogen receptors, proliferative activity marker Ki-67 and inhibitor of apoptosis Bcl-2 was performed. Results. In the group of patients without preoperative hormonal treatment progesterone receptors were expressed in 76,4±6,8% of the nuclei, estrogen receptors - in 32,8±2,6%. In the group of patients after treatment with ulipristal acetate there was a significant decrease of progesterone receptor expression – 36,8±1,28% (p 0,05) . Bcl-2 in the control group was found in 65,4±7,2% cells, in leiomyoma after treatment there was a significant decrease of bcl-2 – 42,6±3,2% (p <0, 05). In leiomyomas without hormonal treatment Ki-67 was determined in 11,8% of the nuclei of smooth muscle cells, and in leiomyomas after ulipristal acetate – in 7,2% leiomyoma cells. Conclusions. In patients after three months of ulipristal acetate treatment there was a significant decrease of expression of progesterone receptor, bcl-2, and Ki-67. Taken together these data evidence reduced action of progesterone on leiomyoma cells, induction of apoptosis and decreased proliferation processes that may cause involution of fibroids. Citation: Kuryk EG, Litvak EO, Chabrat BV, Lysenko BM. [Immunohistochemical characteristic of myoma tissue in patients with uterine leiomyoma after treatment with ulipristal acetate]. Morphologia. 2015;9(3):42-7. Russia

Influence of citric acid and water on thermoplastic wheat flour/poly(lactic acid) blends. I: Thermal, mechanical and morphological properties

Wheat flour was plasticized with glycerol and compounded with poly(lactic acid) in a one-step twin-screw extrusion process in the presence of citric acid with or without extra water. The influence of these additives on process parameters and thermal, mechanical and morphological properties of injected samples from the prepared blends, was then studied. Citric acid acted as a compatibilizer by promoting depolymerization of both starch and PLA. For an extrusion without extra water, the amount of citric acid (2 parts for 75 parts of flour, 25 parts of PLA and 15 parts of glycerol) has to be limited to avoid mechanical properties degradation. Water, added during the extrusion, improved the whole process, minimizing PLA depolymerization, favoring starch plasticization by citric acid and thus improving phases repartition

Influence of citric acid on thermoplastic wheat flour/poly (lactic acid) blends. II. Barrier properties and water vapor sorption isotherms

The effects of citric acid on wheat flour/glycerol/poly(lactic) acid (PLA) blends prepared by one-step twin-screw extrusion have been studied to improve barrier properties of starch based materials. A series of injected samples were produced from prepared compounds with varying ratio (0-20 part) of citric acid. The effects of citric acid on the water vapor permeability, oxygen permeability and water solubility in the film were then investigated. The barrier properties results proved that citric acid behaves as compatibilizing agent between starch and PLA phases for ratios between 0 and 10 parts. When the added amount exceeds 10 parts, CA acted as a plasticizer and/or promoted the hydrolysis of the starch glycosidic bonds

Cystamine/cysteamine rescues the dopaminergic system and shows neurorestorative properties in an animal model of Parkinson's disease.

The neuroprotective properties of cystamine identified in pre-clinical studies have fast-tracked this compound to clinical trials in Huntington's disease, showing tolerability and benefits on motor symptoms. We tested whether cystamine could have such properties in a Parkinson's disease murine model and now provide evidence that it can not only prevent the neurodegenerative process but also can reverse motor impairments created by a 6-hydroxydopamine lesion 3weeks post-surgery. Importantly, we report that cystamine has neurorestorative properties 5weeks post-lesion as seen on the number of nigral dopaminergic neurons which is comparable with treatments of cysteamine, the reduced form of cystamine used in the clinic, as well as rasagiline, increasingly prescribed in early parkinsonism. All three compounds induced neurite arborization of the remaining dopaminergic cells which was further confirmed in ex vivo dopaminergic explants derived from Pitx3-GFP mice. The disease-modifying effects displayed by cystamine/cysteamine would encourage clinical testing

Metabolism

Background: Cardiovascular disease is the leading cause of deaths in nonalcoholic steatohepatitis (NASH) patients. Mouse models, while widely used for drug development, do not fully replicate human NASH nor integrate the associated cardiac dysfunction, i.e. heart failure with preserved ejection fraction (HFpEF). To overcome these limitations, we established a nutritional hamster model developing both NASH and HFpEF. We then evaluated the effects of the dual peroxisome proliferator activated receptor alpha/delta agonist elafibranor developed for the treatment of NASH patients. Methods: Male Golden Syrian hamsters were fed for 10 to 20 weeks with a free choice diet, which presents hamsters with a choice between control chow diet with normal drinking water or a high fat/high cholesterol diet with 10% fructose enriched drinking water. Biochemistry, histology and echocardiography analysis were performed to characterize NASH and HFpEF. Once the model was validated, elafibranor was evaluated at 15 mg/kg/day orally QD for 5 weeks. Results: Hamsters fed a free choice diet for up to 20 weeks developed NASH, including hepatocyte ballooning (as confirmed with cytokeratin-18 immunostaining), bridging fibrosis, and a severe diastolic dysfunction with restrictive profile, but preserved ejection fraction. Elafibranor resolved NASH, with significant reduction in ballooning and fibrosis scores, and improved diastolic dysfunction with significant reduction in E/A and E/E' ratios. Conclusion: Our data demonstrate that the free choice diet induced NASH hamster model replicates the human phenotype and will be useful for validating novel drug candidates for the treatment of NASH and associated HfpEF

"About François", retour sur une pièce de théâtre sur François Abou Salem (1)

Entre fin octobre et décembre 2018 puis en novembre 2019, la création d’une pièce de théâtre - About François - a eu lieu au Théâtre National Palestinien El-Hakawati de Jérusalem-Est (TNP). Elle constitue un événement à plusieurs titres : elle porte sur l’artiste de théâtre François Gaspar dit Abou Salem (1951-2011), co-fondateur de la compagnie El-Hakawati, aujourd’hui le Théâtre National Palestinien. Il s’agit de la première œuvre créée sur ce metteur en scène et directeur de théâtre depuis..