Les cellules épendymaires sont des cellules multiciliées qui tapissent les parois de toutes les cavités du cerveau. Une fois différenciées, ces cellules ne se divisent plus au cours de la vie. Le battement de ces multiples cils motiles joue un rôle important pour maintenir un flux constant de liquide cérébrospinal à travers toutes les cavités cérébrales. Les cellules épendymaires assurent également des fonctions critiques d’échanges moléculaires avec le liquide cérébrospinal. Dans son ensemble, l’implication des cellules épendymaires et de leurs cils motiles s’avère d’une importance majeure dans le maintien des circuits neuraux ainsi que dans le fonctionnement plus global du cerveau. Récemment, une nouvelle caractéristique des cellules épendymaires a été identifiée ; elles font partie d’un microenvironnement appelé une « niche » centrée autour de cellules souches neurales dans le cerveau du rongeur adulte. Ces cellules souches neurales adultes sont capables de produire de nouveaux neurones qui migreront vers le bulbe olfactif des rongeurs adultes. Concernant leur origine, il a été montré que les cellules épendymaires multiciliées dérivent des cellules souches neurales durant les stades tardifs embryonnaires. Ces mêmes cellules souches peuvent d’ailleurs donner naissance à la plupart des différents types de cellules du cerveau. Cependant, les mécanismes par lesquels les cellules souches décident de leur destin cellulaire restent largement méconnus. Dans ce projet, nous étudions quel type de division donne naissance à des cellules épendymaires et nous nous intéressons également au lignage épendymaire. Nos données suggèrent que les cellules épendymaires ne migrent pas après leur dernière division et qu’elles restent à proximité de l’endroit où elles ont été produites. Chose particulièrement intéressante, nous montrons que les cellules épendymaires peuvent être générées par division symétrique ou asymétrique. Nos résultats révèlent aussi que les cellules souches neurales embryonnaires se divisent de manière asymétrique pour donner naissance à la fois à une celluleépendymaire et à une cellule souche neurale adulte. Ces données viennent s’ajouter à la connaissance actuelle que nous avons du développement du cerveau. De plus, elles pourraient contribuer à ouvrir de nouvelles perspectives et stratégies thérapeutiques pour soigner les maladies neurodégénératives à beaucoup plus long terme. / Ependymal cells are multiciliated cells lining the walls of all brain cavities. Once they are mature, they do not divide during life. Their motile ciliary beating endorses a crucial role in maintaining a proper flow of cerebrospinal fluid throughout all brain cavities. Ependymal cells also ensure critical molecular exchanges of the cerebrospinal fluid. On the whole, the involvement of ependymal cells and their multiple motile cilia in the maintenance of the neural circuits and more globally in the well-functioning of the entire brain have proven paramount. More recently, a new characteristic of ependymal cells has been brought to light. Namely, they are part of a microenvironment so called a “niche” surrounding adult neural stem cells in the adult rodent brain. Noteworthy, these adult neuralstem cells are capable of producing new neurons that will migrate to the olfactory bulb of rodents. In terms of their origin, it was shown that multiciliated ependymal cells derive from neural stem cells during late embryonic stages. Besides, the same stem cells can give rise to most cell types of the brain. However, little is known about how fate-decision is made in neural stem cells. In this project, we tackle more particularly how multiciliated ependymal cells arise from the neural stem cells. Most specifically, we address the type of celldivision and the ependymal cell lineage. We find that ependymal cells are not migrating subsequent to their last division, but rather stay where they were first produced. Most interestingly, they can be generated through both symmetric and asymmetric cell division. We also show that embryonic neural stem cells divide asymmetrically to give rise to both an ependymal cell and an adult stem cell. We are confident that these data bring major new insights in the current understanding of neural development. Additionally, these findingscould contribute in opening new therapeutic perspectives and strategies to cure neurodegenerative diseases in a much longer term.

Recent comparative studies have shown that bird brains, although small, have a high processing capacity. The brains of parrots and songbirds have higher neuronal densities than brains of mammals; especially large parrots and corvids compete with or even outnumber primates by the number of telencephalic neurons. However, the processing capacity of the avian brain appears to differ significantly between various phylogenetic lineages. Basal groups such as galliform birds have much lower absolute numbers of neurons and lower neuronal densities than songbirds and parrots. In this Master thesis, I used the isotropic fractionator to determine numbers of neurons and non-neural cells in specific brain regions in 19 species of hornbills (Bucerotiformes), woodpeckers (Piciformes) and coraciiform birds (Coraciiformes). The brains of hornbills and woodpeckers (but not coraciiform birds) have numbers of neurons comparable to that of songbirds and parrots and significantly more neurons than equivalently sized brains of pigeons (Columbiformes) and galliform birds (Galliformes). In the crown groups, we can observe similar trends such as a higher degree of encephalization, a proportionally larger telencephalon and increasing percentage of telencephalic neurons. On the contrary, in pigeons and galliform birds, we can...

<p>Transforming growth factor β-activated kinase 1 (TAK1), a hub kinase at the convergence of multiple signaling pathways, is critical to the development of the central nervous system and has been found to play a role in cell death and apoptosis. TAK1 may have the potential to elucidate mechanisms of cell cycle and neurodegeneration. The Belecky-Adams laboratory has aimed to study TAK1 and its potential roles in cell cycle by studying its role in chick retinal development as well as its possible implication in the progression of diabetic retinopathy (DR). Chapter 3 includes studies that explore TAK1 in a study in chick retinal development and TAK1 in in vitro studies in retinal microglia. Using the embryonic chick, immunohistochemistry for the activated form of TAK1 (pTAK1) showed localization of pTAK1 in differentiated and progenitor cells of the retina. Using an inhibitor or TAK1 activite, (5Z)-7-Oxozeaenol, in chick eye development showed an increase in progenitor cells and a decrease in differentiated cells. This study in chick suggests TAK1 may be a critical player in the regulation of the cell cycle during retinal development. Results from experimentation in chick led to studying the potential role of TAK1 in inflammation and neurodegeneration. TAK1 has previously been implicated in cell death and apoptosis suggesting that TAK1 may be a critical player in inflammatory pathways. TAK1 has been implicated in the regulation of inflammatory factors in different parts of the CNS but has not yet been studied specifically in retina or in specific retinal cells [3, 4]. Chapter 2 includes studies from the Belecky-Adams laboratory of in vitro work with retinal microglia. Retinal microglia were treated with activators and the translocation to the nucleus of a downstream factor of TAK1 was determined: NF-kB. Treatment of retinal microglia in the presence of activators with TAKinib, an inhibitor of TAK1 activation, revealed that TAK1 inhibition reduces the activation of downstream NF-kB. Together this data suggests that TAK1 may be implicated in various systems of the body and further studies on its mechanisms may help elucidate potential therapeutic roles of the kinase.</p>

Coupling of cells to biomaterials is a prerequisite for most biomedical applications; e.g., neuroelectrodes can only stimulate brain tissue in vivo if the electric signal is transferred to neurons attached to the electrodes’ surface. Besides, cell survival in vitro also depends on the interaction of cells with the underlying substrate materials; in vitro assays such as multielectrode arrays determine cellular behavior by electrical coupling to the adherent cells. In our study, we investigated the interaction of neurons and glial cells with different electrode materials such as TiN and nanocolumnar TiN surfaces in contrast to gold and ITO substrates. Employing single-cell force spectroscopy, we quantified short-term interaction forces between neuron-like cells (SH-SY5Y cells) and glial cells (U-87 MG cells) for the different materials and contact times. Additionally, results were compared to the spreading dynamics of cells for different culture times as a function of the underlying substrate. The adhesion behavior of glial cells was almost independent of the biomaterial and the maximum growth areas were already seen after one day; however, adhesion dynamics of neurons relied on culture material and time. Neurons spread much better on TiN and nanocolumnar TiN and also formed more neurites after three days in culture. Our designed nanocolumnar TiN offers the possibility for building miniaturized microelectrode arrays for impedance spectroscopy without losing detection sensitivity due to a lowered self-impedance of the electrode. Hence, our results show that this biomaterial promotes adhesion and spreading of neurons and glial cells, which are important for many biomedical applications in vitro and in vivo.

Biomaterials employed for neural stimulation, as well as brain/machine interfaces, offer great perspectives to combat neurodegenerative diseases, while application of lab-on-a-chip devices such as multielectrode arrays is a promising alternative to assess neural function in vitro. For bioelectronic monitoring, nanostructured microelectrodes are required, which exhibit an increased surface area where the detection sensitivity is not reduced by the self-impedance of the electrode. In our study, we investigated the interaction of neurons (SH-SY5Y) and glial cells (U-87 MG) with nanocolumnar titanium nitride (TiN) electrode materials in comparison to TiN with larger surface grains, gold, and indium tin oxide (ITO) substrates. Glial cells showed an enhanced proliferation on TiN materials; however, these cells spread evenly distributed over all the substrate surfaces. By contrast, neurons proliferated fastest on nanocolumnar TiN and formed large cell agglomerations. We implemented a radial autocorrelation function of cellular positions combined with various clustering algorithms. These combined analyses allowed us to quantify the largest cluster on nanocolumnar TiN; however, on ITO and gold, neurons spread more homogeneously across the substrates. As SH-SY5Y cells tend to grow in clusters under physiologic conditions, our study proves nanocolumnar TiN as a potential bioactive material candidate for the application of microelectrodes in contact with neurons. To this end, the employed K-means clustering algorithm together with radial autocorrelation analysis is a valuable tool to quantify cell-surface interaction and cell organization to evaluate biomaterials’ performance in vitro

Le système nerveux est composé de milliards de connexions synaptiques qui forment des réseaux complexes à la base de la communication dans le cerveau. Dès lors, contrôler la localisation, le type et le nombre des synapses est un défi considérable au cours du développement du système nerveux. Étonnamment, la production de connexions synaptiques est démesurée de façon à ce que beaucoup plus de synapses soient formées au cours du développement que ce qui est maintenu chez l’adulte. Ces connexions surnuméraires sont en compétition pour l’innervation d’une même cellule cible ce qui mène au maintien de certaines terminaisons nerveuses et à l’élimination de d’autres. Ces processus de compétition et d’élimination sont grandement façonnés par l’activité du système nerveux et l’expérience sensorielle de manière à ce que les terminaisons qui montrent la meilleure activité sont favorisées alors que les synapses mal adaptées sont éliminées. Jusqu’à récemment, les mécanismes et les types cellulaires responsables de l’élimination synaptique étaient inconnus. Les études de la dernière décennie montrent que les cellules gliales jouent un rôle clé dans l’élimination de synapses. Cependant, il demeure inconnu si les cellules gliales peuvent décoder les niveaux d’activité des terminaisons en compétition, ce qui est un déterminant majeur de l’issue de la compétition synaptique. De plus, il n’est pas connu si les cellules gliales sont capables de réguler l’activité synaptique des terminaisons, ce qui pourrait influencer l’issue de l’élimination synaptique. Ceci est d’un intérêt particulier puisqu’il est connu que les cellules gliales interagissent activement avec les neurones, détectent et modulent leur activité dans plusieurs régions du système nerveux mature. Par conséquent, l'objectif de cette thèse était d'étudier la capacité des cellules gliales à interagir avec les terminaisons nerveuses en compétition pour l'innervation d’une même cellule cible. Nous avons donc analysé la capacité des cellules gliales à décoder l’activité des terminaisons, à réguler leur activité synaptique et à influencer le processus de l’élimination synaptique au cours du développement du système nerveux. Pour cette fin, nous avons profité de la jonction neuromusculaire, un modèle simple et le bien caractérisé, et nous avons combiné l’imagerie Ca2+ des cellules gliales, un rapporteur fiable de leur activité avec des enregistrements synaptiques de jonctions neuromusculaires poly-innervées de souriceaux. Dans la première étude, nous montrons que les cellules gliales détectent et décodent l'efficacité synaptique des terminaisons nerveuses en compétition. L’activité des cellules gliales reflète la force synaptique de chaque terminaison nerveuse et l'état de la compétition synaptique. Ce décodage est médié par des récepteurs purinergiques gliaux fonctionnellement distincts et les propriétés intrinsèques des cellules gliales. Nos résultats indiquent que les cellules gliales décodent la compétition synaptique et, par conséquent, sont favorablement positionnées pour influencer son issue. Dans la seconde étude, nous montrons que les cellules gliales régulent différemment la plasticité synaptique de terminaisons en compétition. De manière dépendante du Ca2+, les cellules gliales induisent une potentialisation persistante de l’activité de la terminaison forte alors qu’elles n’ont que peu d’effets sur la terminaison faible. Bloquer l'activité gliale altère la plasticité des terminaisons in situ et se traduit par un retard de l'élimination des synapses in vivo. Ainsi, nous décrivons un nouveau mécanisme par lequel les cellules gliales, non seulement renforcent activement la terminaison forte, mais influencent aussi la compétition et l'élimination. Dans l'ensemble, ces études sont les premières à démontrer que les cellules gliales sont activement impliquées dans la modulation de l'activité synaptique des terminaisons en compétition ainsi que dans la régulation de l'élimination synaptique et la connectivité neuronale. / The nervous system is composed of billions of synaptic connections forming complex networks that define the basis of neuronal communication in the brain. The control of the localization, type and number of synapses is a considerable challenge during development of the nervous system. Surprisingly, there is an excessive production of synaptic connections so that many more synapses are formed during developmental stages than what is maintained in the adult. A process of competition and elimination then occurs during which connections are in competition for the innervation of the same target cell. These processes of competition and elimination are greatly shaped by activity and sensory experience. Nerve terminals that show the best activity are favoured, while weak and poorly adapted synapses are eliminated. Until recently, the mechanisms and the cell types responsible for the elimination of supernumerary connections were unknown. Studies from the last decade identified glial cells as major players in synapse elimination. However, it remains unknown whether glial cells are able to decode the levels of synaptic activity of competing terminals, which is a major determinant of the outcome of synaptic competition. Moreover, it is unknown whether glial cells are able to regulate synaptic activity, which could influence the outcome of synapse elimination. This is especially relevant because it is known that glial cells actively interact with neurons, detect and modulate their activity in many regions of the nervous system. Therefore, the goal of this thesis was to study the ability of glial cells to interact with terminals competing for the innervation of the same target cell. We tested the ability of glial cells to decode the activity nerve terminals, regulate their synaptic activity and influence the process of synapse elimination during development of the nervous system. For this purpose, we took advantage of the neuromuscular junction, a simple and well-characterized model, and used simultaneous Ca2+-imaging of glial cells, a reliable reporter of their activity and synaptic recordings of dually-innervated neuromuscular junctions from newborn mice. In the first study, we report that single glial cells detect and decode the synaptic efficacy of competing nerve terminals. Activity of single glial cells reflects the synaptic strength of each competing nerve terminal and the state of synaptic competition. This deciphering is mediated by functionally segregated purinergic receptors and intrinsic properties of glial cells. Our results indicate that glial cells decode ongoing synaptic competition and, hence, are poised to influence its outcome. In the second study, we show that glial cells differentially regulate the synaptic plasticity of competing terminals. In a Ca2+-dependent manner, glial cells induce a long lasting synaptic potentiation of strong but not weak terminals. Preventing glial activity alters the plasticity of terminals in situ and delays synapse elimination in vivo. Thus, we describe a novel mechanism by which glial cells, not only actively reinforce the strong input but regulate synapse competition and elimination. As a whole, these studies are the first to demonstrate that glial cells are actively involved in the modulation of synaptic activity of competing terminals as well as in the regulation of synapse elimination and neuronal connectivity.

Entre os PRRs (receptores de reconhecimento padrão), NOD-like receptors (NLRs), tal como NOD2, são responsáveis pela detecção intracelular de muramil dipeptídeo (MDP); padrão molecular associado a patógeno (PAMP), encontrado no peptidoglicano (PGN) de praticamente todas bactérias GRAM positiva e negativa. Após o reconhecimento e estimulação por MDP, NOD2 recruta diretamente a serina-treonina quinase RIPK2, uma proteína adaptadora importante na ativação de NF?B mediada por NOD2. A expressão de NOD2 foi descrita em macrófagos e em outras células. Além disso, trabalhos anteriores indicaram que PRRs desempenham papel crucial na ativação de células gliais da medula espinal, na indução e manutenção da dor inflamatória crônica e dor neuropática. No presente estudo, avaliamos o papel de NOD2 na modulação da sensibilidade à dor, focando sua importância na ativação de células da glia da medula espinal, bem como a sua via de sinalização (RIPK2) e liberação de citocinas pró-nociceptivas, como o fator de necrose tumoral alfa (TNF-?), interleucina-6 (IL-6) e interleucina-1 beta (IL-1?). Os resultados demonstram que camundongos selvagens tratados com MDP, apresentaram diminuição no limiar nociceptivo mecânico (pico entre 3 e 5 horas) comparado com o grupo controle (veículo), retornando ao basal após 48 horas. Além disso, camundongos NOD2-/- , RIPK2-/- , TNFR1/2-/- e IL-6 -/- tratados com MDP não diferiram o limiar nociceptivo mecânico, comparado com seus respectivos grupos controle (veículo). Entretanto, camundongos TNFR1- /- , CCR2-/- , TLR4-/- , MyD88-/- e TRIF-/- tratados com MDP, apresentaram diminuição no limiar nociceptivo mecânico similar aos camundongos selvagens tratados com MDP. Adicionalmente, o pré-tratamento de camundongos selvagens com IL-1ra, propentofilina, minociclina, fluorocitrato e SB 203580 inibiu o desenvolvimento da hipersensibilidade mecânica induzida por MDP. Estes dados sugerem que a ativação do sensor intracellular NOD2 esta presente em células da glia da medula espinal e estimula a ativação das vias de sinalização RIPK2 e p38 MAPK com subsequente produção de IL-1?, IL-6 e TNF?, por uma via de sinalização independente de TLR4, MyD88 e TRIF. Finalmente, estes mecanismos contribuem para o processo de hipersensibilidade mecânica durante a neuropatia periférica e representam uma nova abordagem para elucidar os mecanismos envolvidos na fisiopatologia da dor crônica. / Among PRRs (pattern recognition receptors), NOD-like receptors (NLRs), such as NOD2 are responsible by intracellular detection of muramyl dipeptide (MDP); pathogen-associated molecular pattern (PAMP) found in the peptidoglycan (PGN) from virtually all gram positive and gram negative bacteria. Upon recognition and stimulation by MDP, NOD2 recruits directly the receptor-interacting serine/threonine-protein kinase 2 (RIPK2), an adaptor protein important in the NOD2-mediated NF?B activation. The expression of NOD2 has been described in macrophages and other cells. Moreover, previous work has indicated that PRRs play a crucial role in the activation of spinal cord glial cells, in the induction and maintenance of chronic inflammatory and neuropathic pain. In the present study, we aimed to evaluate the role of NOD2 in the modulation of pain sensitivity, focusing on its importance in the activation of spinal cord glial cells, as well as its signaling pathway (RIPK2) and release of pro-nociceptive cytokines, such as tumour necrosis factor-alpha (TNF-?), interleukin-6 (IL-6) and interleukin-1beta (IL-1?). The results demonstrate that WT mice treated with MDP showed a decrease in mechanical nociceptive threshold (peak 3 to 5 hours) compared with the control group (vehicle), returning to the base line after 48 hours. Furthermore, NOD2-/- , RIPK2-/- , TNFR1/2-/- and IL-6 -/- mice treated with MDP did not differ the mechanical nociceptive threshold compared with their respective control groups (vehicle). However, TNFR1-/- , CCR2-/- , TLR4-/- , MyD88-/- and TRIF-/- mice treated MDP, showed a decrease in mechanical nociceptive threshold similar to WT mice treated with MDP. In addition, the pretreatment of WT mice with IL-1ra, propentofylline, minocycline, fluorocitrate and SB 203580 inhibited the development of mechanical hypersensitivity induced by MDP. These data suggest that activation of the intracellular sensor NOD2 present in spinal cord glial cells stimulates the activation of RIPK2 and p38 MAPK signaling pathways and subsequent production of IL-1?, IL-6 and TNF?, in a TLR4-, MyD88- and TRIF-independent signaling pathway. Finally, these mechanisms contribute to the process of mechanical hypersensitivity during peripheral neuropathy and represent a novel approach for elucidating the mechanisms underlying pathophysiology of chronic pain.

Neureguline (NDF, Heregulin, GGF ARIA, oder SMDF) sind EGF-ähnliche, extrazelluläre Signalmoleküle, die mit transmembranären Tyrosinkinaserezeptoren der ErbB-Familie interagieren. Neuregulin-1/ErbB Signale steuern während der Embryonalentwicklung und im adulten Organismus vielfältige zelluläre Prozesse, wie z. B. Proliferation, Migration und Differenzierung. In der vorliegenden Arbeit wurde die Rolle des Neuregulin-1/ErbB Signalsystems in der Entwicklung von Neuralleistenzellen und sich daraus entwickelnden Komponenten des peripheren Nervensystems (sympathisches Nervensystem und periphere Glia) untersucht. Neuregulin-1 Signale werden in Neuralleistenzellen und ihren Gliaderivaten durch ErbB2/ErbB3 Rezeptor-Heterodimere übertragen. Mit Hilfe von Mäusen mit gezielter Mutation (knock-out) des Neuregulin-1, ErbB2 oder ErbB3 Gens wurde gezeigt, daß Neuregulin-1/ErbB2/3 Signale die Migration sympathogener Neuralleistenzellen steuern. Mutante Tiere entwickeln daher eine hochgradige Hypoplasie des sympathischen Nervensystems. Neuregulin-1 Signale kontrollieren darüber hinaus die Entwicklung von Schwann Zellen. Die Unterbrechung des Neuregulin-1/ErbB2/3 Signalwegs in mutanten Mäusen führt zum Verlust von Schwann Zellen während der Embryogenese. Es wurde außerdem gezeigt, daß der Transkriptionsfaktor Sox10 die Expression von ErbB3 in Neuralleistenzellen kontrolliert. Sox10 und ErbB3 Mutanten besitzen daher übereinstimmende Defekte in der Neuralleistenzellentwicklung. Neben den ErbB3-abhängigen Funktionen von Sox10 wurde eine ErbB3-unabhängige Schlüsselfunktion von Sox10 bei der Differenzierung von Neuralleistenzellen zu peripherer Glia identifiziert. Das Neuregulin-1/ErbB2/3 Signalsystem und der Transkriptionsfaktor Sox10 besitzen also gemeinsam zentrale Funktionen in der Entwicklung peripherer Glia, steuern diesen Prozess aber über unterschiedliche Mechanismen und während unterschiedlicher Entwicklungsphasen. Sox10, ErbB2 und ErbB3 mutante Mäuse entwickeln neben dem Verlust von Schwann Zellen eine sekundäre Degeneration begleitender sensorischer und motorischer Neurone. Dies zeigt, daß periphere Glia Signale generiert, die essentiell sind für Integrität und Überleben begleitender Neurone. / Neuregulins (NDF, heregulin, GGF ARIA, or SMDF) are EGF-like growth and differentiation factors that signal through tyrosine kinase receptors of the erbB family. The neuregulin-1 proteins and their receptors play essential roles during embryonic development and in the adult. Functions of the neuregulin/erbB signaling system in developing neural crest cells and their derivatives (sympathetic nervous system, peripheral glial cells) were analyzed in mice with targeted mutations in the erbB2, erbB3, or neuregulin-1 genes. All three mutations cause severe hypoplasia of the primary sympathetic ganglion chain, and migration of sympathogenic neural crest cells to their target sites, where they differentiate into sympathetic neurons, depends on neuregulin-1 and its receptors. Neuregulin-1 signals are also essential for the development of Schwann cells. As a consequence, mice with targeted mutations in the neuregulin-1/erbB signaling system completely lack Schwann cells. Moreover, the HMG-box transcription factor sox10 is shown to control expression of erbB3 in neural crest cells. In accordance, sox10 and erbB3 mutant mice share phenotypes in the developing neural crest. Additionally, a novel, erbB3-independent developmental function of sox10 was identified: Sox10 is a key regulator for glial fate determination in undifferentiated neural crest cells. Thus, the transcription factor sox10 and the neuregulin-1/erbB signalling system both serve critical functions during development of peripheral glial cells. However, they act via different cellular mechanisms and during different developmental stages. At later developmental stages lack of peripheral glial cells in sox10, erbB2 and erbB3 mutant mice results in a severe degeneration of sensory and motor neurons. The comparison of the mutant phenotypes demonstrates, that peripheral glial cells generate essential signals for the survival and maintenance of accompanying neurons.

Entre os PRRs (receptores de reconhecimento padrão), NOD-like receptors (NLRs), tal como NOD2, são responsáveis pela detecção intracelular de muramil dipeptídeo (MDP); padrão molecular associado a patógeno (PAMP), encontrado no peptidoglicano (PGN) de praticamente todas bactérias GRAM positiva e negativa. Após o reconhecimento e estimulação por MDP, NOD2 recruta diretamente a serina-treonina quinase RIPK2, uma proteína adaptadora importante na ativação de NF?B mediada por NOD2. A expressão de NOD2 foi descrita em macrófagos e em outras células. Além disso, trabalhos anteriores indicaram que PRRs desempenham papel crucial na ativação de células gliais da medula espinal, na indução e manutenção da dor inflamatória crônica e dor neuropática. No presente estudo, avaliamos o papel de NOD2 na modulação da sensibilidade à dor, focando sua importância na ativação de células da glia da medula espinal, bem como a sua via de sinalização (RIPK2) e liberação de citocinas pró-nociceptivas, como o fator de necrose tumoral alfa (TNF-?), interleucina-6 (IL-6) e interleucina-1 beta (IL-1?). Os resultados demonstram que camundongos selvagens tratados com MDP, apresentaram diminuição no limiar nociceptivo mecânico (pico entre 3 e 5 horas) comparado com o grupo controle (veículo), retornando ao basal após 48 horas. Além disso, camundongos NOD2-/- , RIPK2-/- , TNFR1/2-/- e IL-6 -/- tratados com MDP não diferiram o limiar nociceptivo mecânico, comparado com seus respectivos grupos controle (veículo). Entretanto, camundongos TNFR1- /- , CCR2-/- , TLR4-/- , MyD88-/- e TRIF-/- tratados com MDP, apresentaram diminuição no limiar nociceptivo mecânico similar aos camundongos selvagens tratados com MDP. Adicionalmente, o pré-tratamento de camundongos selvagens com IL-1ra, propentofilina, minociclina, fluorocitrato e SB 203580 inibiu o desenvolvimento da hipersensibilidade mecânica induzida por MDP. Estes dados sugerem que a ativação do sensor intracellular NOD2 esta presente em células da glia da medula espinal e estimula a ativação das vias de sinalização RIPK2 e p38 MAPK com subsequente produção de IL-1?, IL-6 e TNF?, por uma via de sinalização independente de TLR4, MyD88 e TRIF. Finalmente, estes mecanismos contribuem para o processo de hipersensibilidade mecânica durante a neuropatia periférica e representam uma nova abordagem para elucidar os mecanismos envolvidos na fisiopatologia da dor crônica. / Among PRRs (pattern recognition receptors), NOD-like receptors (NLRs), such as NOD2 are responsible by intracellular detection of muramyl dipeptide (MDP); pathogen-associated molecular pattern (PAMP) found in the peptidoglycan (PGN) from virtually all gram positive and gram negative bacteria. Upon recognition and stimulation by MDP, NOD2 recruits directly the receptor-interacting serine/threonine-protein kinase 2 (RIPK2), an adaptor protein important in the NOD2-mediated NF?B activation. The expression of NOD2 has been described in macrophages and other cells. Moreover, previous work has indicated that PRRs play a crucial role in the activation of spinal cord glial cells, in the induction and maintenance of chronic inflammatory and neuropathic pain. In the present study, we aimed to evaluate the role of NOD2 in the modulation of pain sensitivity, focusing on its importance in the activation of spinal cord glial cells, as well as its signaling pathway (RIPK2) and release of pro-nociceptive cytokines, such as tumour necrosis factor-alpha (TNF-?), interleukin-6 (IL-6) and interleukin-1beta (IL-1?). The results demonstrate that WT mice treated with MDP showed a decrease in mechanical nociceptive threshold (peak 3 to 5 hours) compared with the control group (vehicle), returning to the base line after 48 hours. Furthermore, NOD2-/- , RIPK2-/- , TNFR1/2-/- and IL-6 -/- mice treated with MDP did not differ the mechanical nociceptive threshold compared with their respective control groups (vehicle). However, TNFR1-/- , CCR2-/- , TLR4-/- , MyD88-/- and TRIF-/- mice treated MDP, showed a decrease in mechanical nociceptive threshold similar to WT mice treated with MDP. In addition, the pretreatment of WT mice with IL-1ra, propentofylline, minocycline, fluorocitrate and SB 203580 inhibited the development of mechanical hypersensitivity induced by MDP. These data suggest that activation of the intracellular sensor NOD2 present in spinal cord glial cells stimulates the activation of RIPK2 and p38 MAPK signaling pathways and subsequent production of IL-1?, IL-6 and TNF?, in a TLR4-, MyD88- and TRIF-independent signaling pathway. Finally, these mechanisms contribute to the process of mechanical hypersensitivity during peripheral neuropathy and represent a novel approach for elucidating the mechanisms underlying pathophysiology of chronic pain.

Le gène split end (spen) est impliqué dans de nombreuses voies de signalisation et processus biologiques. Durant ma thèse j'ai étudié le rôle de spen dans la mort cellulaire au cours du développement de la rétine de la Drosophile. L'œil de Drosophile est composé de centaines d'unités appelées ommatidies. Chaque ommatidie est composée de huit photorécepteurs entourés de cellules accessoires comprenant quatre cellules cônes et deux cellules pigmentaires primaires, ainsi que douze cellules interommatidiales. Les cellules interommatidiales adoptent une structure hexagonale parfaitement régulière. Des cellules interommatidiales en excès doivent être éliminées par apoptose au cours du développement. J'ai montré que la modulation de spen modifiait radicalement le patron des cellules interommatidiales. L'inactivation de spen conduit à un défaut de cellules interommatidiales alors que sa surexpression entraîne un excès de ces cellules. Ces résultats témoignent d’un rôle anti-apoptotique de spen. Nous avons aussi montré que la perte des cellules interommatidiales dans un contexte mutant pour spen pouvait être entièrement sauvée en exprimant la protéine p35 connue pour bloquer l'activité des caspases. Comme spen est exprimé de manière ubiquitaire, nous avons cherché à déterminer dans quelles cellules spen jouait son rôle de régulateur de la mort cellulaire. Grâce à une analyse clonale, nous avons pu montrer que c'est au niveau des cellules cônes que spen agit. L'inactivation de spen dans les autres cellules accessoires de l'œil n'influence pas la mort des cellules interommatidiales. Nous avons en outre, montré que spen avait un rôle dans la formation des soies de chaque ommatidie. Ces travaux mettent en évidence un rôle de spen dans le contrôle de la mort cellulaire des cellules interommatidiales dans les cellules cônes. Nos résultats montrent, par ailleurs, que spen serait requis pour le relarguage du facteur de survie Spitz (le ligand activateur de la voie EGF) à partir des cellules cônes. En parallèle, nous avons étudiés le rôle de survie de spen dans un modèle neurodégénératif. Nous avons montré que spen était nécessaire dans les cellules gliales pour la résistance au stress oxydatif. De manière intéressante, nous avons trouvé que l'inactivation de spen dans la glie diminuait l'activité de la voie de signalisation NOTCH. Cette résistance pourrait se faire via la modulation de gènes antioxydants. De manière générale, nos travaux démontrent un rôle du gène split ends dans la survie cellulaire. Ce facteur agit de manière non-autonome à partir des cellules supports de différents organes. / In metazoan, the successful development of many organs requires the elimination of supernumerary cells by apoptosis. For example, the elimination of about two thousand interommatidial cells (IOCs) during Drosophila eye development allows the precise rearrangement of ommatidia in a perfect hexagonal array. Maximal apoptosis occurs during pupal life and the remaining IOCs differentiate into secondary and tertiary pigment cells. The precise removal of unwanted IOCs requires coordinated activation of Notch (pro-death) and EGF (pro-survival) pathways. IOCs undergoing apoptosis express the IAP inhibitor Hid, which leads to the activation of initiator and effector caspases. However, the mechanisms that coordinate the death and survival pathways for timed and precise IOC removal are poorly understood.Here, we report that spen encodes a nuclear protein expressed in the pupal eye that is required for IOC survival. We showed that the inhibition of spen, by either RNAi or in spen mutant clones resulted in disorganized ommatidia with missing IOCs. Moreover, overexpression of spen leads to extra IOCs. These results indicate that spen expression promotes IOC survival during eye development. Importantly blocking apoptosis prevents the loss of IOC in a spen mutant retina. Spen is a protein known to be ubiquitous in tissue during development. Indeed, we have shown using an enhancer trap line that spen is expressed in all the cells in the eye pupal disk. To better understand where spen is acting from in this tissue to regulate cell death, we performed a clonal analysis. We found that the inactivation of spen in the cone cells was causing the loss of IOC, indicating that spen is required non-autonomously in cone cell for IOC survival. In parallel we have shown that the inactivation of spen was disrupting eye bristles morphology. Even if studies discuss the role of bristles in the regulation of developmental apoptosis in this context, our clonal analysis excluded this possibility. Furthermore, we found that spitz, the EGFR ligand, accumulate in cone cells upon spen inactivation. Our current hypothesis is that spen is likely to be required for the release of Spitz from the cone cells in order to active the survival signaling pathway EGFR in the IOCs. Also, we examined the protective role of spen in a chemical model of Parkinson disease (paraquat treatment). We showed that the glial expression of spen is protective in this context, which suggest against that spen acts non-autonomously. Interestingly we found that the inactivation of spen in glia downregulates the Notch signaling pathway. Spen is likely to be a key factor integrating cues from different signaling pathways to promote cell survival.