Motor neuron disorders are a heterogeneous group of diseases characterized by the selective degeneration of motor neurons leading to muscle wasting and atrophy. Amyotrophic Lateral Sclerosis (ALS) is the most common amongst these disorders and is characterized by the selective loss of both upper and lower motor neurons in the brain and spinal cord. 20% of familial cases of ALS are caused by mutations in the Cu, Zn-superoxide dismutase gene (SOD1), a ubiquitously expressed enzyme responsible for scavenging superoxide radicals. The exact mechanisms underlying mutant SOD1-mediated neurotoxicity are unknown. Misfolded mutant SOD1 accumulates in the cytosol and mitochondrial intermembrane space (IMS) indicating the involvement of unfolded protein responses in ALS pathogenesis. Unfolded protein responses (UPRs) are complex signal transduction cascades which detect perturbations in protein folding and couple them to the expression of protein quality control machinery thereby allowing individual compartments to adapt to stress. In the cytosol, this study has shown that HspB8 was upregulated by SOD1 mutants, where it induced the clearance of aggregates by macroautophagy. This is a protective mechanism, as overexpression of HspB8 suppressed mutant-SOD1 mediated toxicity. In contrast, HspB8 mutants were impaired in macroautophagy and are toxic to NSC-34 cells. The mechanisms for the IMS-UPR have not been previously identified. To address this issue, a model for the accumulation of misfolded mutant SOD1 within the IMS was created and candidate proteins involved in protein quality control within the IMS were explored at the transcriptional level and at the level of protein expression. Preliminary results revealed some possible candidates that may have a role in the adaptation to mitochondrial stress. Interestingly, increased mitophagy was also found in IMS-G93A expressing cells, advocating the central role of macroautophagy in eliminating protein aggregates and damaged mitochondria in SOD1-FALS.

One-dimensional neural networks comprised of large numbers of Integrate-and-Fire neurons have been widely used to model electrical activity propagation in neural slices. Despite these efforts, the vast majority of these computational models have no analytical solutions. Consequently, my Ph.D. research focuses on a specific class of homogeneous Integrate-and-Fire neural network, for which analytical solutions of network dynamics can be derived. One crucial analytical finding is that the traveling wave acceleration quadratically depends on the instantaneous speed of the activity propagation, which means that two speed solutions exist in the activities of wave propagation: one is fast-stable and the other is slow-unstable. Furthermore, via this property, we analytically compute temporal-spatial spiking dynamics to help gain insights into the stability mechanisms of traveling wave propagation. Indeed, the analytical solutions are in perfect agreement with the numerical solutions. This analytical method also can be applied to determine the effects induced by a non-conductive gap of brain tissue and extended to more general synaptic connectivity functions, by converting the evolution equations for network dynamics into a low-dimensional system of ordinary differential equations. Building upon these results, we investigate how periodic inhomogeneities affect the dynamics of activity propagation. In particular, two types of periodic inhomogeneities are studied: alternating regions of additional fixed excitation and inhibition, and cosine form inhomogeneity. Of special interest are the conditions leading to propagation failure. With similar analytical procedures, explicit expressions for critical speeds of activity propagation are obtained under the influence of additional inhibition and excitation. However, an explicit formula for speed modulations is difficult to determine in the case of cosine form inhomogeneity. Instead of exact solutions from the system of equations, a series of speed approximations are constructed, rendering a higher accuracy with a higher order approximation of speed.

The worm Caenorhabditis elegans is a well-studied model organism in numerous aspects of its biology. This small free living nematode has less than 1,000 cells, but shows clear conservation in both signaling and behavior to mammals in aspects of appetite control. This is of importance to humans, where failure of appetite control is a major factor in the unprecedented obesity epidemic that we see today. In general, worm behavior reflects its internal nutritional state and the availability and quality of food. Specifically, worms show a behavioral state that mimics aspects of the mammalian behavioral satiety sequence, which has been termed satiety quiescence. We have used locomotion tracking and Hidden Markov Model analysis to identify worm behavioral state over time, finding quiescence along with the established worm locomotive behaviors roaming and dwelling. Using this analysis as well as more conventional cell biology and genetic approaches we have further investigated satiety signaling pathways. We have found that the neuron ASI is a major center of integration of signals regarding the internal nutritional state of the worms as well as the nutritional content of its environment. Our results show that cGMP causes levels of the TGFβ ligand to be increased in fasted worms, which is then released and binds to its receptor on the RIM and RIC neurons. This signaling connects nutritional state to behavioral response, promoting the sleep-like behavioral state satiety quiescence. Additionally, we have begun a candidate approach examining several other groups of signaling molecules for potential roles in satiety quiescence signaling including cannabinoids, multidrug resistance proteins, and neuropeptides. The result of this investigation is a better understanding of mechanisms of satiety quiescence signaling as well as a new tool that provides highly quantitative, unbiased, and automated data to aid in our ongoing work.

We present a multi-layer multi-column computational model of neocortex that is built based on the activity and connections of known neuronal cell types and includes activity-dependent short term plasticity. This model, a network of spiking neurons, is validated by showing that it exhibits activity close to biology in terms of several characteristics: (1) proper laminar flow of activity; (2) columnar organization with focality of inputs; (3) low-threshold-spiking (LTS) and fast-spiking (FS) neurons function as observed in normal cortical circuits; and (4) different stages of epileptiform activity can be obtained with either increasing the level of inhibitory blockade, or simulation of NMDA receptor enhancement. The aim of this research is to provide insight into the fundamental properties of vertical and horizontal inhibition in neocortex and their influence on epileptiform activity. The developed model was used to test novel ideas about modulation of inhibitory neuronal types in a developmentally malformed cortex. The novelty of the proposed research includes: (1) design and implementation of a multi-layer multi-column model of the cortex with multiple neuronal types and short-time plasticity, (2) modification of the Izhikevich neuron model in order to model biological maximum firing rate property, (3) generating local field potential (LFP) and EEG signals without modeling multiple neuronal compartments, (4) modeling several known conditions to validate that the cortex model matches the biology in several aspects,(5) modeling different abnormalities in malformed cortex to test existing and to generate novel hypotheses.

Planar cell polarity (PCP) is a developmental signaling mechanism that establishes a polarity within the plane of an epithelium. PCP has been shown to play a role in guiding numerous neurodevelopmental processes such as convergent extension, neuron migration, and axon pathfinding. Certain commissural neurons in the dorsal spinal cord make a series of guidance decisions en route to the brain: first, a ventral projection along the D-V axis, followed by a midline crossing, and after exiting the floorplate, a dorso-anterior turn along the A-P axis. Here, we provide in vivo evidence that the axons of the Commissural Primary Ascending (CoPAs) neurons in zebrafish require the PCP genes fzd3a, vangl2, and scribble for rostral pathfinding both before and after crossing the midline. Dorsoventral guidance of CoPA axons is unaltered in fzd3a, vangl2, and scribble mutants, suggesting that the PCP signaling pathway only controls A-P guidance of CoPAs. Our results have provided evidence for two potential non- mutually exclusive models: (i) A-P axon guidance is achieved by cell-autonomous Wnt-Frizzled signaling or that (ii) A-P axon guidance is achieved by non-cell-autonomous PCP signaling in the neuroepithelial environment. The single-cell nature of the CoPA axon system allows for simple genetic manipulation and visualization, which will potentially elucidate the validity of either model. Scribble (Scrib), a member of the LAP family, plays a critical role in establishing and regulating cell polarization in epithelia and during cell migration. In zebrafish, Scrib mutants have defects in convergent extension (CE) cell movements and facial branchiomotor neuron (FBMN) migration. Despite our understanding of Scrib’s genetic role in neurodevelopment, little is known about the subcellular localization of endogenous Scrib in vivo during CE and FBMN migration. We have generated a monoclonal antibody against the C-terminus of zebrafish Scrib and have shown that this antibody is specific against endogenous Scrib in both western blot and immunocytochemical applications. Confocal microscopy of Scrib immunocytochemistry shows that at various developmental stages, Scrib distinctly localizes to basolateral membranes of non polarized epithelium, to the membrane in mesodermal cells undergoing CE, and to the membrane of migrating FBMNs. Furthermore, the distribution of Scrib puncta along membranes of FBMN- FBMN contact is significantly altered in the PCP mutant pk1b. Further application of our newly generated Scrib antibody will potentially lead to new insight on Scrib’s role in neurodevelopment.

In the present study, we have examined the expression of protease-activated receptors (PARS) and characterized their signaling pathways in rabbit gastric muscle cells. Immunoblot analysis revealed expression of PARl and PAR2 but not PAR3 or PAR4 in smooth muscle. The PARl agonist TFLLR activated Gq, G12, and Gi3, but not Gil, Gi2, G13, Gs or Gz, whereas the PAR2 agonist SLIGRL activated Gq, G13, Gil, and Gi2, but not Gi3, G12, Gs, or Gz. Both PARl and PAR2 agonists stimulated PI hydrolysis and Rho kinase activity and inhibited cAMP formation. PAR1-stimulated PI hydrolysis was abolished in cells expressing Gαq minigene, but was not affected in cells expressing Gαi minigene or in cells treated with pertussis toxin (PTx). PAR2-stimulated PI hydrolysis was partially inhibited in cells expressing Gαq or Gαi minigene and in cells treated with PTx. PAR1- and PAR2-stimulated Rho kinase activity was abolished in cells expressing Gα12 or Gα13 minigene, respectively. Both PARl and PAR2 agonists induced a transient initial contraction that was selectively blocked by the inhibition of PI hydrolysis with U73122 and MLC kinase activity with ML-9. PAR1-induced sustained contraction was preferentially inhibited by the PKC inhibitor bisindolylmaleimide and to a minor extent by the Rho kinase inhibitor Y27632, whereas PAR2-induced sustained contraction was preferentially inhibited by Y27632. Activation of both PARl and PAR2 induced MLC20 phosphorylation, whereas phosphorylation of MYPTl and CPI-17 are receptor-specific: only PARl induced CPI-17 phosphorylation and only PAR2 induced MYPTl phosphorylation.Activation of PARl and PAR2 also induced IκBα degradation and NF-κB activation; the effects were abolished by the blockade of RhoA activity by Clostridium botulinum C3 exoenzyme suggesting NF-κB is downstream of RhoA. PAR1- and PAR2-stimulated Rho kinase activity was significantly augmented by the inhibitors of PKA (PKI), IKK2 (IKKIV), or NF-κB (MG132), and in cells expressing dominant negative mutants of IKK (IKK(K44A), IκBα (IκBα (S32A/S36A)), or phosphorylation-deficient RhoA (RhoA(S188A)). In addition, activation of PARl induced Gα12 phosphorylation, which was abolished by bisindolylmaleimide, suggests that phosphorylation was mediated by PKC derived from the activation of RhoA. Only PAR1-stimulated Rho kinase activity was significantly augmented by the PKC inhibitor. The effect of PKC inhibitor was additive to that of the PKA inhibitor.

La fragmentation de l'appareil de Golgi représente un des changements les plus précoces et les plus répandus dans les maladies neurodégénératives. Afin de comprendre les mécanismes moléculaires de ces changements, j'ai étudié deux modèles expérimentaux de maladie du neurone moteur. 1. Les souris pmn (progressive motor neuronopathy) : Celles-ci sont atteintes d'une forme très grave de dégénérescence des neurones moteurs et des défauts moléculaires sont liés à une mutation faux-sens d'une protéine localisée au niveau du Golgi, la chaperonne des tubulines TBCE, identifiée par (Martin, Jaubert et al. 2002; Schaefer, Schmalbruch et al. 2007). Au cours de ma thèse, nous avons identifié des anomalies importantes du Golgi dans les neurones moteurs lombaires de souris pmn et déterminé leur relevance fonctionnelle ainsi que les mécanismes moléculaires. D'après les immunomarquages et la modélisation 3D des membranes, la fragmentation et l'atrophie du Golgi dans les neurones lombaires moteurs pmn ressemblent à celles rapportées dans la SLA et se produit dans des cinétiques similaires. Les analyses en microcopie électronique montrent que l'empilement des citernes golgiennes est progressivement remplacé par des petites vésicules. Les analyses biochimiques révèlent : 1/ une redistribution cytosolique des protéines d'arrimage tel que GM130, 2/ une diminution des protéines β-COP et 3/ une augmentation considérable des protéines golgiennes d'amarrage v-SNARE GS15 et GS28 contrôlant la fusion des vésicules. / Fragmentation of the Golgi apparatus represents one of the earliest and most constant pathological changes in neurodegenerative diseases. To understand the molecular mechanisms of these changes I investigated two experimental models of motor neuron diseases. 1. pmn mice with progressive motor neuronopathy. The pmn mice were chosen since they suffer from a very aggressive form of motor neuron degeneration and since their molecular defects represents a missense mutation in a Golgi-localized tubulin chaperone TBCE, as shown by previous (Martin et al 2002, Schäfer et al 2007). In the last years, we identified severe Golgi abnormalities in motor neurons of pmn mice and dissected out their functional relevance and molecular mechanisms. According to immunolabelings and 3D membrane modelings, Golgi fragmentation and atrophy in lumbar pmn motor neurons resembled those reported in human ALS and proceeded with similar kinetics. Electron microscopy illustrated that Golgi cisternae were progressively transformed into small vesicles. Biochemical analyses revealed : 1/ a cytosolic redistribution of tethering factor such as GM130, 2/ a decrease in β-COP protein level and 3/ a massive increase in the Golgi v-SNARE proteins GS15 and GS28 controlling vesicle fusion. These pathological changes were due to loss of TBCE expression since they could be rescued by transgenic expression of wildtype TBCE but not mimicked by sciatic nerve axotomy. They involved defective dynamics of Golgi-derived microtubules rather than accumulation of misfolded tubulins as shown by the differential effects of TBCE-depletion, Nocodazole and a folding-incompetent tubulin mutant.

Les récepteurs du glutamate de type AMPA (rAMPA) sont les récepteurs ionotroniques responsables de la majeure partie des courants excitateurs rapides lors de la transmission synaptique dans le système nerveux central. Le nombre de rAMPA stabilisés à la synapse est responsable en partie de l’intensité de la transmission synaptique et de nombreux phénomènes de plasticité synaptique. Les rAMPA se répartissent en trois populations en équilibre dynamique: les récepteurs intracellulaires, les récepteurs extra-synaptiques, et les récepteurs synaptiques stabilisés au niveau de la densité post-synaptique. L’implication des protéines transmembranaires régulatrices des rAMPA (TARP) dans la stabilisation des rAMPA est établie, et repose au moins en partie sur la liaison de la protéine TARP γ-2 avec la protéine d’échafaudage PSD-95. Dans l’hippocampe, siège de nombreux phénomènes de plasticité, l’isoforme γ-8 est particulièrement enrichie. La TARP γ-8 a pour particularité de posséder un domaine C-terminal plus long que son homologue γ-2 et de s’exprimer au niveau synaptique et extra-synaptique. Mon travail de thèse à consisté à étudier les mécanismes moléculaires mis en jeu dans la régulation de la liaison des protéines TARP γ-2 et γ-8 avec la protéine PSD-95, ainsi que l’implication respective des deux isoformes dans la régulation de la mobilité latérale des rAMPA. Les résultats majeurs de cette étude sont : a) l’interaction entre γ-2 et PSD-95 est régulée par la longueur apparent du domaine C-terminal de γ-2 modulée par la phosphorylation; b) γ-8 lie PSD-95 dans les compartiments synaptiques et extra-synaptiques, toutefois cette interaction n’est pas corrélée avec une immobilisation des rAMPA. Ces résultats suggèrent que γ-2 et γ-8 jouent des rôles bien distincts dans l’adressage des rAMPA à la synapse. / AMPA type glutamate receptors (AMPARs) are ionotropic receptors responsible for most excitatory transmission in the central nervous system. The number of stabilized AMPARs in front of glutamate release sites determines in large part the strength of synaptic transmission and variation in this number is thought to underlie numerous forms of synaptic plasticity. AMPARs are present in three main subcellular pools between which they are in a dynamic equilibrium by processes of trafficking: intracellular receptors, extrasynaptic receptors, and synaptic receptors stabilized at the postsynaptic density (PSD). Transmembrane AMPAR regulatory proteins (TARPs) are known to be implicated in AMPAR stabilization at the synapse through the interaction of TARP γ-2/8 with the scaffolding protein PSD-95. In the hippocampus, a structure exhibiting various synaptic plasticity patterns, γ-8 is the most abundant TARP. This isoform is characterized by a longer C-terminal fragment than γ-2 and a synaptic and extrasynaptic localization. During my Ph.D, I studied the molecular mechanisms involved in the regulation of TARP γ-2 and γ-8 binding to PSD-95 and their respective roles in regulating AMPAR lateral mobility. The main results are: a) γ-2 interaction with PSD-95 is regulated by the apparent length of its C-terminus domain that is modulated by phosphorylation; b) γ-8 binds PSD-95 in synaptic and extrasynaptic compartment however this interaction is not correlated with AMPAR immobilization. Altogether, those results suggest that those two TARP isoforms have independent functional roles.

Les pathologies du système somatosensoriel, appelées neuropathies sensitives périphériques, touchent environ 3 millions de personnes en France et causent des déficits sensoriels multiples. Parmi elles, les douleurs neuropathiques post traumatiques sont les plus fréquentes et sont souvent chroniques et résistantes aux traitements actuels. Une lésion nerveuse périphérique induit des réponses cellulaires permettant la survie et la régénération de ces neurones. Les ganglions rachidiens dorsaux (DRG) contiennent une variété de neurones sensitifs qui transmettent les stimuli somatiques. Suite à une blessure du nerf périphérique les neurones sensitifs s'adaptent à un nouvel environnement pour réussir leur élongation axonale. Parmi les mécanismes cellulaires conduisant à une croissance neuritique améliorée, il a été démontré qu'une lésion primaire in vivo du nerf augmente la régénération axonale suite à une deuxième lésion. In vitro, les neurones qui ont été conditionnés par le premier traumatisme montrent une croissance neuronale plus rapide et plus élonguée appelée croissance régénérative. L'élasticité est un paramètre déterminant des propriétés mécaniques de la membrane cellulaire. Elle donne des informations importantes sur la santé et la fonction de la cellule. Le microscope à force atomique (AFM) est devenu de nos jours un outil commun pour l'imagerie à haute résolution de matériaux biologiques puisque les cellules vivantes peuvent être imagées dans leurs conditions physiologiques. En plus du rôle des propriétés élastiques dans le processus de régénération, l'organisation structurale des tissus est en grande partie déterminante du degré et de la direction de croissance et du mouvement cellulaire. Le guidage de la croissance par la modification des surfaces ou « patterning » est possible avec la technique de « microcontact printing » qui permet la conception de circuits de protéines avec des géométries bien définies. Les protéines de la matrice extracellulaire. Dans la première partie de la thèse nous avons mis en évidence les propriétés mécaniques de la membrane de neurones sensitifs issus de DRG de souris adultes suite à une lésion du nerf sciatique gauche. Les neurones sensitifs conditionnés montrent un mode de croissance neuritique plus rapide et plus élonguée, moins de branchements neuritiques et plus de souplesse membranaire des somas et des cônes de croissance. Dans un deuxième volet du travail nous avons réussi à normaliser la pousse régénérative et l'activité électrique des neurones sensitifs et motoneurones spinaux en utilisant le patterning des protéines d'adhésion cellulaire (ECM) dans le but d'imiter la croissance longitudinale in vivo. / Peripheral nerve injuries lead to paralysis, anesthesia and lack of autonomic control of the affected body areas. The trauma results in loss of motor and sensory functions conveyed by the involved nerves. This process is referred to as Wallerian degeneration; it creates a microenviroment in the injury site that favors neurites regrowth. The increased intrinsic growth capacity of injured peripheral neurons is manifested experimentally by the conditioning lesion paradigm. Axotomy of a peripheral neuron previous to the test lesion, ‘‘primes'' the neuron, switches it on to a regenerative state and, thus, it will regenerate faster after receiving the second injury. Mechanical interactions play a key role in many processes associated with neuronal growth and development. Membrane cytoskeleton elasticity is a determining parameter of membrane mechanical properties and provides important information toward the health and function of the cell. For this reason the first objective of this thesis was to understand the conditioning injury effects on both morphology and rheological properties of live sensory neurons cell bodies and growth cones, using particularly the atomic force microscopy, and to correlate this to eventual modifications in the composition of the cytoskeletal proteins. In addition to the role of cell elastic properties and mechanical sensing in the regeneration process, the structural organization of tissues plays a major part in deciding the degree and direction of tissue growth and cell movement. The ability to guide cells and their outgrowth by modifying surfaces is possible with the microcontact printing technique which enables the design of protein pathways with experimentally defined geometries. Therefore, the second objective of the thesis was to modulate the regenerative growth of dorsal root ganglia sensory neurons and spinal motoneurons using cell adhesion proteins in order to physically mimic the in vivo longitudinal axonal growth. We used the extracellular matrix (ECM) proteins, ideal biomolecules for printing as they can guide in vitro the cellular adhesion, differentiation, migration. The patterning allowed us to normalize neurite elongation and electrical activity of sensory neurons before and after conditioning lesion.

Desde que surgiu, o computador vem sendo utilizado na modelagem de fenômenos em todas as áreas do conhecimento. Em neurociências, a modelagem computacional é utilizada para descrever, reproduzir e fazer previsões sobre o comportamento dos diferentes componentes do sistema nervoso. Assim como em outras áreas das ciências, este procedimento tem-se mostrado eficiente no estudo e aprimoramento das teorias a respeito da função cerebral. Com o crescente aumento do poder computacional, maiores e mais detalhados modelos podem ser construídos com um grau de realismo biológico cada vez maior. Neste trabalho, apresentamos modelos computacionais biologicamente plausíveis de neurônios corticais do sistema somestésico primário. Os modelos foram construídos com base no formalismo de Hodgkin-Huxley para a implementação de canais iônicos e na técnica de compartimentalização de Rall para modelar sua extensão espacial. Os parâmetros foram ajustados a partir de resultados experimentais 'in vivo' e 'in vitro' com neurônios, retirados da literatura. Os resultados das simulações mostraram que os modelos são biologicamente aceitáveis e de qualidade superior a de outros modelos construídos anteriormente, possibilitando a construção de modelos de redes neuronais em larga escala mais precisos. / Since its appearance, the computer has been used to model phenomena in all areas of knowledge. In neuroscience, computer modeling is used to describe, reproduce and predict behaviors of different components of the nervous system. As well as in other areas of sciences, this procedure has been shown to be efficient in the study and improvement of theories on brain function. With the increasing power of computers, larger and more detailed models can be constructed with an increasing degree of biological realism. In this work, we present biologically plausible computer models of cortical neurons from the primary somatosensory system. The models have been implemented based on the Hodgkin-Huxley formalism for ionic channels and the Rall´s compartmental technique for spatial extent. The parametrs have been adjusted based on in vivo and in vitro experimental results taken from the literature. Simulation results have shown that the models are biologically acceptable and of superior quality in comparison with previous models, allowing the construction of more precise large-scale neuronal network models.