Induction of early Purkinje cell dendritic differentiation by thyroid hormone requires RORα

<p>Abstract</p> <p>Background</p> <p>The active form (T₃) of thyroid hormone (TH) controls critical aspects of cerebellar development, such as migration of postmitotic neurons and terminal dendritic differentiation of Purkinje cells. The effects of T₃on early dendritic differentiation are poorly understood.</p> <p>Results</p> <p>In this study, we have analyzed the influence of T₃on the progression of the early steps of Purkinje cell dendritic differentiation in postnatal day 0 organotypic cerebellar cultures. These steps include, successively, regression of immature neuritic processes, a stellate cell stage, and the extension of several long and mature perisomatic protrusions before the growth of the ultimate dendritic tree. We also studied the involvement of RORα, a nuclear receptor controlling early Purkinje cell dendritic differentiation. We show that T₃treatment leads to an accelerated progression of the early steps of dendritic differentiation in culture, together with an increased expression of RORα (mRNA and protein) in both Purkinje cells and interneurons. Finally, we show that T₃failed to promote early dendritic differentiation in <it>staggerer </it>RORα-deficient Purkinje cells.</p> <p>Conclusions</p> <p>Our results demonstrate that T₃action on the early Purkinje cell dendritic differentiation process is mediated by RORα.</p

Identification of genes involved in ceramide-dependent neuronal apoptosis using cDNA arrays

BACKGROUND: Ceramide is important in many cell responses, such as proliferation, differentiation, growth arrest and apoptosis. Elevated ceramide levels have been shown to induce apoptosis in primary neuronal cultures and neuronally differentiated PC 12 cells. RESULTS: To investigate gene expression during ceramide-dependent apoptosis, we carried out a global study of gene expression in neuronally differentiated PC 12 cells treated with C(2)-ceramide using an array of 9,120 cDNA clones. Although the criteria adopted for differential hybridization were stringent, modulation of expression of 239 genes was identified during the effector phase of C(2)-ceramide-induced cell death. We have made an attempt at classifying these genes on the basis of their putative functions, first with respect to known effects of ceramide or ceramide-mediated transduction systems, and then with respect to regulation of cell growth and apoptosis. CONCLUSIONS: Our cell-culture model has enabled us to establish a profile of gene expression during the effector phase of ceramide-mediated cell death. Of the 239 genes that met the criteria for differential hybridization, 10 correspond to genes previously involved in C(2)-ceramide or TNF-α signaling pathways and 20 in neuronal disorders, oncogenesis or more broadly in the regulation of proliferation. The remaining 209 genes, with or without known functions, constitute a pool of genes potentially implicated in the regulation of neuronal cell death

Synapto-protective drugs evaluation in reconstructed neuronal network

Chronic neurodegenerative syndromes such as Alzheimer’s and Parkinson’s diseases, or acute syndromes such as ischemic stroke or traumatic brain injuries are characterized by early synaptic collapse which precedes axonal and neuronal cell body degeneration and promotes early cognitive impairment in patients. Until now, neuroprotective strategies have failed to impede the progression of neurodegenerative syndromes. Drugs preventing the loss of cell body do not prevent the cognitive decline, probably because they lack synapto-protective effects. The absence of physiologically realistic neuronal network models which can be easily handled has hindered the development of synapto-protective drugs suitable for therapies. Here we describe a new microfluidic platform which makes it possible to study the consequences of axonal trauma of reconstructed oriented mouse neuronal networks. Each neuronal population and sub-compartment can be chemically addressed individually. The somatic, mid axon, presynaptic and postsynaptic effects of local pathological stresses or putative protective molecules can thus be evaluated with the help of this versatile “brain on chip” platform. We show that presynaptic loss is the earliest event observed following axotomy of cortical fibers, before any sign of axonal fragmentation or post-synaptic spine alteration. This platform can be used to screen and evaluate the synapto-protective potential of several drugs. For instance, NAD+ and the Rho-kinase inhibitor Y27632 can efficiently prevent synaptic disconnection, whereas the broad-spectrum caspase inhibitor zVAD-fmk and the stilbenoid resveratrol do not prevent presynaptic degeneration. Hence, this platform is a promising tool for fundamental research in the field of developmental and neurodegenerative neurosciences, and also offers the opportunity to set up pharmacological screening of axon-protective and synapto-protective drugs

Wallerian-Like Degeneration of Central Neurons After Synchronized and Geometrically Registered Mass Axotomy in a Three-Compartmental Microfluidic Chip

Degeneration of central axons may occur following injury or due to various diseases and it involves complex molecular mechanisms that need to be elucidated. Existing in vitro axotomy models are difficult to perform, and they provide limited information on the localization of events along the axon. We present here a novel experimental model system, based on microfluidic isolation, which consists of three distinct compartments, interconnected by parallel microchannels allowing axon outgrowth. Neurons cultured in one compartment successfully elongated their axons to cross a short central compartment and invade the outermost compartment. This design provides an interesting model system for studying axonal degeneration and death mechanisms, with a previously impossible spatial and temporal control on specific molecular pathways. We provide a proof-of-concept of the system by reporting its application to a well-characterized experimental paradigm, axotomy-induced Wallerian degeneration in primary central neurons. Using this model, we applied localized central axotomy by a brief, isolated flux of detergent. We report that mouse embryonic cortical neurons exhibit rapid Wallerian-like distal degeneration but no somatic death following central axotomy. Distal axons show progressive degeneration leading to axonal beading and cytoskeletal fragmentation within a few hours after axotomy. Degeneration is asynchronous, reminiscent of in vivo Wallerian degeneration. Axonal cytoskeletal fragmentation is significantly delayed with nicotinamide adenine dinucleotide pretreatment, but it does not change when distal calpain or caspase activity is inhibited. These findings, consistent with previous experiments in vivo, confirm the power and biological relevance of this microfluidic architecture

Les mécanismes moléculaires de la dégénérescence axonale dans un contexte de type Dying back pattern

Dans la Maladie d Alzheimer, les neurones meurent par un processus de mort cellulaire programmée, appelé apoptose. Des études in vivo, ont montré que cette étape de destruction somatique, tardive, est précédée de phénomènes précoces de dysfonctionnements synaptiques et de dégénérescences axonales. Néanmoins, en l absence d outils appropriés, leurs modalités et leurs mécanismes moléculaires demeurent encore mal connus. Nous nous proposons d aborder cette problématique en utilisant un nouveau système de culture primaire de neurones appelé microfluidique permettant la compartimentalisation efficace des axones et des somas de neurones issus du système nerveux central. Grâce à cet outil nous mettons en évidence qu un stress appliqué uniquement au niveau du corps cellulaire est suffisant pour induire une dégénérescence axonale. Il semble donc qu un signal de dégénérescence se soit propagé dans l axone depuis le soma. D un point de vue moléculaire, la map kinase JNK impliquée dans les phases précoces de l apoptose est ici le premier signal observé dans l axone, contrôlant la fission des mitochondries et l activation des caspases, qui sont les effecteurs moléculaires de la phase d exécution de l apoptose. De plus, nous avons démontré que le NAD+, un élément clef dans la bioénergétique du neurone, était lui aussi impliqué. Ainsi nos résultats suggèrent que le NAD+ et le programme apoptotique peuvent ensemble participer au devenir de l axone. En conclusion, ce travail s intègre dans la compréhension des mécanismes de la dégénérescence axonale observée dans les maladies neurodégénératives telle que la Maladie d AlzheimerIn Alzheimer's disease, neurons die through a process of programmed cell death, called apoptosis. In vivo studies have shown that this stage of somatic destruction, late, is preceded by phenomena of early synaptic dysfunction and axonal degeneration. However, in the absence of appropriate tools, their methods and their molecular mechanisms remain poorly understood. We propose to tackle this issue using a new system of primary culture of neurons called "microfluidic" for effective compartmentalization of axons and somas of neurons from the central nervous system. With this tool we show that a stress applied only at the cell body is sufficient to induce axonal degeneration. It therefore seems that a signal has spread degeneration in the axon from the soma. From a molecular point of view, the map kinase JNK involved in the early stages of apoptosis is by the first signal observed in the axon, controlling the fission of mitochondria and caspase activation, which are the molecular effectors of the execution phase of apoptosis. Furthermore, we demonstrated that the NAD +, a key element in the bioenergetics of the neuron, was also involved. Thus our results suggest that NAD + and apoptosis can all become involved in the axon. In conclusion, this work fits into understanding the mechanisms of axonal degeneration observed in neurodegenerative diseases such as Alzheimer's diseasePARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

Reconstruction de voies neuro-anatomiques en culture microfluidique pour l étude des mécanismes de dégénérescence transynaptique

Les pathologies neurodégénératives comme la maladie d Alzheimer ou de Parkinson sont caractérisées par un dysfonctionnement neuronal lent conduisant à des altérations synaptiques et de manière ultime à la mort neuronale. De récentes études suggèrent qu au cours du temps, les stigmates pathologiques et la dégénérescence qui affectent initialement des aires restreintes, pourraient s amplifier et se disperser au sein d aires voisines via un chemin préférentiel. Plusieurs possibilités non exclusives pourraient expliquer cette progression: la perte de facteur neurotrophique, une neurotransmission aberrante, la dispersion de protéines aggrégées Dans ma thèse j ai développé un système de culture microfluidique permettant d orienter la croissance axonale afin de reconstruire des réseaux neuronaux orientés modélisant des voies neuroanatomiques. J ai ensuite modélisé une dégénérescence locale aigüe (axotomie corticale) ou une lésion agrégative locale (dépôt focal d A somatique cortical). Ces paradigmes induisent une déconnexion présynaptique précoce au niveau des connexions pré/post-synaptiques (à distance du stress) et m a permis d évaluer le potentiel synapto-protecteur de molécules pharmacologiques. Cette dégénérescence présynaptique induit des perturbations dans les neurones distants connectés non exposés au stress avant la dégénérescence globale du neurone directement ciblé. La genèse d une neurotransmission glutamatergique aberrante pourrait médier ce phénomène via l altération des patrons d activation des récepteurs extrasynaptiques mais aussi synaptiquesPARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

Développement et activité de réseaux de neurones in vitro (enseigner la chimie organique par le jeu)

Ma thèse comporte deux grandes parties, la première en biophysique et la seconde en science de l éducation. La première partie présente des travaux à la frontière entre neurobiologie et microfluidique. Le but de ces travaux est de pouvoir reconstruire et étudier des réseaux complexes de neurones in vitro avec une topologie de connections synaptiques bien contrôlées. Une série de micro-structures mécanique et/ou chimique ont été étudiées pour leur capacité à (i) positionner les corps cellulaires des neurones, (ii) orienter la pousse des neurites, et (iii) différencier les axones des dendrites. Un premier réseau comportant trois populations de neurones connectées en série a été reconstruit à l intérieur d un circuit microfluidique. Ce réseau qui mime la voie perforante de l hippocampe pourra être exploité pour des études en physiologie ou en neuro-dégénerescence. Une méthode entièrement optique de stimulation et d observation de l activité neuronal a été mise au point. Elle ouvre de nouvelles portes pour étudier des processus cognitifs complexes dans des systèmes simplifiés in vitro. La seconde partie de mon travail a permis le développement et l étude de jeux pédagogiques pour l apprentissage de la chimie en licence. Ces jeux, qui peuvent selon les cas remplacer un cours ou une séance d exercices, donnent des résultats prometteurs pour l aide à la compréhension et à la mémorisation de concepts tels que la géométrie des molécules ou la réactivité entre molécules organiques.My PhD is divided in two parts one on biophysic of neuronal networks and one on science of education. The first part present results at the frontier between neurobiology and microfluidic. The overarching goal of this work was to develop tools and methods to build and study complex neuronal networks controlling the topology of synaptic connexions. Micro-patterning techniques with mechanical and/or chemical constraints were explored regarding their capacity to (i) position cell bodies, (ii) orient neurite outgrowth and (iii) polarize neurons. For the first time, a network comprising three different neuronal populations connected in specified directions was reconstructed in a microfluidic device. This network that mimics the perforant pathway of the hippocampus can be used to study physiological rythms or neurodegenerative processes including Alzheimer s disease. A novel and fully optical method is presented to stimulate and record neuronal activity in vitro. It opens new routes to study complex cognitive processes in simplified in vitro systems. The second part of my work present the development and assessment of educational games in chemistry at the undergraduate level. These games that can either be used to replace courses or exercises, seem promising to improve the understanding and memorization of chemistry concepts og geometries of molecules and organic reactivity.PARIS5-Bibliotheque electronique (751069902) / SudocSudocFranceF

The association of interest memantine plus vitamin D on the prevention of the death of neurons subjected to an aggression by the blood

International audienceno abstrac