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Ce travail évalue l’efficacité d’un peptide dérivé d’une protéine TSR dans la réparation des lésions traumatiques de la substance blanche du système nerveux central. Les propriétés neuroprotectrices ont été explorées sur l’activité anti-oxydante et anti-apoptotique. Le NX210 augmente la viabilité des cellules B104 exposées au peroxyde d’oxygène, un des principaux radicaux oxygénés de la réaction secondaire. Lemécanisme anti-apoptotique a été étudié par la mesure de l’activité anti-caspase. Le NX210 inhibe l’activité des caspases 3/7 selon un effet dose dépendant. Les propriétés neuroréparatrices ont été testées sur des modèles de lésions médullaires chez le rat. Dans un modèle de section médullaire, le NX210 stimule précocement la croissance axonale et la fasciculation. Deux molécules impliquées dans la fasciculation, les neurofilaments et la laminine, sont colocalisées au niveau de la repousse. L’efficacité clinique du NX210 a été testée sur un modèle de contusion médullaire. Le poids corporel était précocement et constamment supérieur chez les animaux traités par rapport aux témoins. L’amélioration motrice évaluée en «open-field» comprenait une augmentation de la distance parcourue et une diminution du temps passé dans les cellules centrales. Un score de BBB supérieur à 14 chez les animaux traités signait la restauration de la coordination des mouvements entre les membres thoraciques et les membres pelviens. La normalisation des réflexes pouvait être corrélée à la restitution du contrôle supraspinal, notamment par la repousse des fibres corticospinales, rubrospinales et extéroceptives. L’action sur le recrutement cellulaire a été étudiée sur un modèle de section du corps calleux en immunohistochimie. La réalisation d’une lésion proche de la zone subventriculaire permettait d’explorer le recrutement cellulaire à partir de cette niche à cellules souches. L’absence de cellule NeuN+ tendrait à démontrer l’absence de recrutement neuronal. Le marquage au MBP montrait des débris de myéline à distance du foyer lésionnel liés à des phénomènes de dégénérescence wallérienne ou d’excitoxicité. La présence de cellules GFAP+ etNG2+ sur le site lésionnel témoignait d’un recrutement astrocytaire et oligodendrocytaire. / In this work, we have studied the efficacy of a TSR-derived peptide in white matter repair. Neuroprotective properties were studied using two models of oxidative stress and apoptosis in vitro. NX210 increases cell viability after exposition to H2O2, one the main ROS that take part in the secondary lesion. Anti-oxidant action was mediated by the scavenger property of the molecule and the stimulation of signaling pathway. Anti-apoptotic action was assessed by measuring caspase 3/7 activity. NX210 inhibits caspase 3/7 activity according to a dose effect relation. Neurorepair was assessed using two separate rat models of spinal cord injury (SCI). In the model provided by section of both dorsal funiculi, NX210 stimulates early axonal growth that predominates on sensory fibers and displays a fasciculate organization. At the site of regrowth, neurofilaments were colocalized with laminin, a molecule involved in fasciculation and axonal guidance during embryogenesis. Clinical efficiency was assessed using a contusive model of SCI. Body weight was early and constantly increased in NX210 treated animals as compared to vehicle treated animals. Improvement in locomotor behavior was appraised with the open field tests. Path length was significantly increased while time spent in central cells was constantly decreased in NX210 treated animals. A BBB score above 14 only performed by NX210 treated animals was related to the restoration of coordination between forelimbs and hind limbs. Normalization of reflexes such as paw placement and toe spread in NX210 treated animals could becorrelated to the recovery of supraspinal control. The action on cell recruiting was assessed by immunohistochemistry using a rat model of corpus callosum section. The lesion was performed near the subventricular zone to study cell proliferation and migration from the stem cell niche to the site of injury. The lack of NeuN immunostaining confirmed the absence of neural cells recruitment. Myelin debris identified by MBP immunostaining were located at a distance from the site of injury. GFAP and NG2cells significantly more numerous in NX210 treated animals identified astrocyte and oligodendrocyte recruitment all around the lesion site.

Remyelination describes the regeneration of myelin sheaths, and is considered one of the most promising strategies for improving the prognosis of demyelinating diseases such as multiple sclerosis. Data from animal models and human studies have shown that remyelination can occur extensively in the central nervous system (CNS), leading to functional recovery and axonal protection. However, remyelination does not always proceed to completion, and its failure is associated with progressive neurological disability. Thus, there is clinical need for interventions that can optimise the conditions for remyelination. Recent advances in genomics and animal husbandry have kindled an interest in the microbiome as a means to influence processes throughout the body. Our commensal microbes communicate with host cells at epithelial barriers, stimulate neural and endocrine axes and directly produce a plethora of long-range signalling molecules. Critically, the development and maintenance of the immune system depend on signals from the microbiota, and we know that a well-coordinated immune response is a key determinant of the success of remyelination. This thesis explores how the microbiome can influence CNS remyelination. To do so, I have studied remyelination in three murine models of microbiome alteration. Firstly, long-term oral administration of an antibiotic cocktail was used to deplete the microbiota of adult mice. Following focal demyelination, these mice had deficits in their inflammatory response, clearance of myelin debris and differentiation of new oligodendrocytes from oligodendrocyte progenitor cells (OPCs). Faecal microbial transplant was able to rescue aspects of the inflammatory response and phagocytosis, but not OPC differentiation. Secondly, I looked at remyelination in germ-free (GF) mice following cuprizone-induced demyelina- tion. As with the antibiotics-treated mice, there were deficits in inflammation following demyelination, which tended to peak later than in control mice. Finally, I investigated the potential of a therapeutic probiotic (VSL#3) to improve remyelination in aged mice. In contrast to antibiotic treatment, probiotic administration caused a slight enhancement in the onset of inflammation following focal demyelination. However, there was no significant improvement in OPC differentiation or toluidine blue rank analysis, suggesting these changes in inflammation were not sufficient to positively modulate remyelination. The results from these three studies introduce a significant but previously unconsidered environmental influence on remyelination in the CNS. Whilst the effects are subtle relative to more direct interventions, the microbiome can be manipulated simply and non-invasively, which may provide a useful adjunct to other strategies for optimising remyelination.

L'évaluation non-invasive de la myéline dans la substance blanche du système nerveux central est fondamentale pour le suivi de pathologies telles que la sclérose en plaques. La myéline est majoritairement constituée de lipides et de protéines : du fait des nombreuses interactions dans ces macromolécules, les temps de relaxation transversale sont très courts (T2 < 1 ms), rendant indétectables ces signaux par des séquences conventionnelles. Les méthodes standards d’imagerie par RMN pour la caractérisation de la myéline reposent sur la modélisation des interactions entre les protons aqueux et la structure myélinisée. Néanmoins, la sélectivité et la robustesse de ces méthodes indirectes peuvent être remises en cause. Les séquences à temps d’écho ultracourt (UTE – TE < 1 ms) permettraient de faire l’acquisition directe des signaux issus de la matrice semi-solide de la myéline. Le développement de telles méthodes pour la mise en contraste positif et sélectif de la myéline sur système préclinique est l’objet de cette thèse. La validation de chacune des méthodes a été menée sur modèle murin ex vivo en confrontant des animaux sains et démyélinisés. Les résultats à partir des méthodes UTE montrent une sélectivité significative à la démyélinisation, suggérant l’adéquation de la technique pour l'évaluation de la myéline dans la substance blanche. / Non-invasive evaluation of white matter myelin in the central nervous system is essential for the monitoring of pathologies such as multiple sclerosis. Myelin is essentially composed of lipids and proteins: because of the numerous interactions between these macromolecules, the transverse relaxation times are very short (T2 < 1 ms), and their signals are undetectable using conventional sequences. Standard MRI methods for the characterization of myelin rely on the modeling of the interactions of aqueous protons with myelinated structures. Nonetheless, the selectivity and robustness of such indirect methods are questionable. Ultrashort echo time sequences (UTE – TE < 1 ms) may allow to directly detect the signals arising from the semi-solid spin pool of myelin. The main objective of this thesis consists in developing such methods in order to generate a positive and selective contrast of myelin using a preclinical imaging system. Validation of each method was carried out using an ex vivo murine model by confronting healthy and demyelinated animals. Results show a significant selectivity of the UTE methods to demyelination, suggesting that the technique is promising for white matter myelin monitoring.

Myelinated axons are crucial for rapid information transmission within the central nervous system (CNS). Myelin injury is a common feature of white matter (WM) pathology in a number of disease states, including ischemic stroke. Myelin disruption can lead to a complete failure in saltatory action potential conduction, resulting in devastating neurological deficits. However, the fundamental mechanism of ischemic myelin injury is controversial. Glutamate-mediated excitotoxicity is now recognised as a crucial event in the development of ischemic WM pathology. This thesis investigates the potential mechanisms of glutamate release in central WM and examines the hypothesis that NMDA receptor over-activation mediates ischemic myelin damage. Using glutamate biosensor microelectrodes and FM-dye imaging, I show that axonal depolarisation in the adult corpus callosum evokes rapid vesicular docking in axons, capable of elevating extracellular glutamate concentration. My findings show that vesicular fusion occurs under the myelin sheath in myelinated axons, which supports the existence of a novel synapse between the axon and overlaying myelin. Simulation of ischemia triggered an early and robust rise in optic nerve extracellular glutamate levels. Unexpectedly, a significant component of ischemic glutamate release also originated from axonal vesicular fusion. Together, these findings show that the axon-myelin synapse represents a significant site of excitotoxic injury during ischemia. Resolving prior conflicting results, I show that NMDA receptor antagonists prevent myelin degradation and improve functional recovery when applied for sufficient time to penetrate the sheath. Finally, I identify a fluorescent myelin stain (QNZ-46) which is a negative allosteric modulator of NR2C/D-containing NMDA receptors. QNZ-46 selectively accumulates in myelinated WM regions of the CNS following systemic administration, and is retained following wash-out. As a result, QNZ-46 provides persistent protection during ischemia by preserving myelin structure and improving functional recovery.

Schwann cells are the principal glial cell of the peripheral nervous system and are responsible for axon maintenance, regeneration and increasing saltatory conduction of neurons. Schwann cell differentiation and myelination is mediated by a core network of transcription factors and signalling pathways, which have been divided into two groups; positive and negative regulators. Sox10, NFATc4, Oct6, Krox20 and the ERK 1/2 signalling pathway have been characterised as positive regulators of Schwann cell differentiation and myelination; with Sox10 and Krox20 also playing critical roles in myelin maintenance. On the other hand, transcription factors cJun, Pax3, Id2 and signalling pathways Notch and p38 mitogen activated protein (MAP) kinases (MAPK) have been identified as negative regulators of Schwann cell differentiation and myelin formation. Recently, the HMG transcription factor Sox2 was identified as a negative regulator of Schwann cell myelination in vitro, however its role in Schwann cell myelination in vivo has not yet been studied. This study therefore aimed to examine the role of Sox2 overexpression in Schwann cells and how it effects Schwann cell differentiation and myelination during development and after injury. In addition, we aimed to investigate for the first time the specific role of p38α (the major isoform of p38 MAPK) in Schwann cell myelination in vivo, by generating Schwann cell specific p38α conditional knockout mice. Sox2 is highly expressed in immature Schwann cells, but is downregulated as Schwann cells being to mature and differentiate. This study shows that continued expression of Sox2 during development and after injury, impairs Schwann cell differentiation and myelination by directly downregulating the expression of two core transcription factors; Sox10 and Krox20, as well as myelin proteins, P0 and MBP. In addition, we observe that continued Sox2 expression significantly increases Schwann cell proliferation and maintains Schwann cells in an immature state. Unexpectedly, we also observed that continued Sox2 expression significantly increases the number of macrophages present in the nerves of Sox2 overexpressing mice at both P60 and 21 days post injury. Phenotypically, Sox2 overexpressing mice 6 show signs of a peripheral neuropathy and animals have impaired motor and sensory function. These findings confirm that Sox2 is a negative regulator of Schwann cell myelination and suggests that continued Sox2 expression is sufficient to drive the progressive development of a peripheral nerve disorder which may resemble Charcot-Marie-Tooth type 1 demyelinating neuropathy and congenital hypomyelinating neuropathy. As a negative regulator of Schwann cell myelination, activity of the p38 MAPK pathway has been shown to inhibit myelin formation in vitro and to also induce the Schwann cell injury response; by driving Schwann cell dedifferentiation and demyelination following injury. Here we show that specific removal of the p38α isoform in Schwann cells leads to an increase in myelin thickness at early developmental time-points, along with an elevated expression of myelin proteins, P0 and MBP. Further analysis following nerve injury revealed that removal of p38α results in an initial decrease in Schwann cell demyelination, yet improves axon remyelination at 21 days post injury. These results demonstrate the specific role of p38α in regulating Schwann cell myelination, and how it could be a direct therapeutic target for improving nerve repair after injury.

La synthèse de la gaine de myéline périphérique par les cellules de Schwann dans le système nerveux périphérique est sous le contrôle de plusieurs facteurs de transcription incluant Egr2 qui est aussi impliqué dans la maintenance de la myéline périphérique. Nous avons développé une nouvelle méthode pour étudier l'implication de différents gènes dans la maintenance de la myéline périphérique en utilisant des siRNA pour inactiver des gènes cibles in vitro et in vivo. Nous avons utilisé des siRNA modifiés pour inhyber les gènes candidats sans utiliser de réactif de transfection. Ces siRNA sont d'abord utilisés sur des co-cultures organotypiques de ganglions rachidiens postérieurs d'embryons de rat et aussi en les injectant dans le nerf sciatique de rat adulte. Nous avons montré que les siRNA contrôles n'entrainent pas de démyelinisation significative aussi bien sur les co-cultures que in vivo après l'injection directe dans le nerf sciatique. Nous avons alors montré que les siRNA anti Egr2 régulent à la baisse l'expression de ces gènes cibles d'environ 60%. En plus, le traitement avec les siRNA anti Egr2 entrainent une dégradation de la gaine de myéline périphérique dans les co-cultures. L'injection des siRNA anti-Egr2 dans le nerf sciatique de rat adulte induit une demyelinisation rapide et significative marquée par la perte de l'expression de MPZ (myelin protein zero) dans la zone d'injection montrée par les expériences immunohistochimiques (microscopie optique) et par l'observation directe de la démyelinisation à l’épon sur des sections transversales de nerf sciatique ( microscopie optique et électronique). Ces résultats confirment ceux précédents impliquant Egr2 dans la maintenance active de la myéline qui avaient été obtenus par des expériences de « Knockout » conditionnels sur des souris. Des injections des siRNA anti-dicer ont induit de manière similaire la démyelinisation de nerf sciatique, établissant que l’expression de Dicer dans les nerfs périphériques adultes est nécessaire pour la maintenance correcte de la myéline. Nos résultats constituent une preuve de concept de l’utilisation de siRNA pour comprendre les mécanismes moléculaires de la maintenance de la myéline in vivo et les gènes induisant la démyelinisation dans les nerfs périphériques. / The synthesis of the myelin sheath by Schwann cells in the peripheral nervous system is under the control of several transcription factors including Egr2, which was shown to be also involved in the maintenance of peripheral myelin. We set up a new method to study the involvement of various genes in peripheral myelin maintenance, using small interfering RNAs (siRNAs) to silence target genes in vitro and in vivo. We used modified self-delivery siRNAs to silence candidate genes without using transfection reagents. These siRNAs were first used on organotypic co-cultures of dorsal root ganglia from embryonic rat and then injected into the sciatic nerves of adult rats. We showed that control non-targeting siRNAs did not induce significant demyelination either in co-cultures or in vivo, after direct injection in sciatic nerves. We then showed that anti-Egr2 siRNAs down-regulated in vitro their target gene expression by ~60%. In addition, treatment with anti-Egr2 siRNAs resulted in abnormalities of the myelin sheaths in co-cultures. The injection of anti-Egr2 siRNAs in the sciatic nerves of adult rats induced a significant and rapid demyelination, as shown by the loss of Myelin Protein Zero expression in the injected area on immunohistochemistry experiments (optic microscopy) and by direct evidence of demyelination on epon-embedded transversal sections of sciatic nerves (optic and electron microscopy). These results confirm previous data involving Egr2 in active myelin maintenance, which were obtained using conditional knockout experiments in mice. Injections of anti-Dicer siRNAs similarly induced sciatic nerve demyelination, establishing that Dicer expression in adult peripheral nerves is necessary for proper myelin maintenance. Our results constitute a proof of concept for the use of self-delivery siRNAs to investigate the molecular mechanisms of myelin maintenance in vivo and also to induce a gene-driven demyelination “on demand” in peripheral nerves.

La production physiologique continue de cellules myélinisantes dans le système nerveux (SN) de mammifère offre de nouvelles perspectives thérapeutiques. Lors d’une atteinte de la myéline, une régénération endogène impliquant la génération d’oligodendrocytes s’engage. Ce processus repose sur la mobilisation de progéniteurs oligodendrocytaires parenchymateux et de progéniteurs de la zone sous-ventriculaire (SVZ). Cette réparation ne permet cependant pas une récupération fonctionnelle systématique. Nos travaux ont pour but d’identifier les facteurs qui contrôlent les différentes étapes de régénération. Ils révèlent une réexpression du CNTF et une surexpression des héparans sulfates protéoglycanes (HSPGs) suite à une démyélinisation du corps calleux. Ces changements de l’environnement péri-lésionnel régulent positivement le processus de remyélinisation. Nous avons montré un impact direct de l’expression post-lésionnelle du CNTF sur la mobilisation des deux sources cellulaires. Différents tests in vitro ont identifié le CNTF comme facteur chémoattractant pour ces cellules. Nos données montrent également que des modifications de sulfatation des héparans sulfates (HS) protéoglycanes contrôlées par la N-désacétylase-Sulfotransférase 1 des cellules du lignage oligodendrocytaire s’établissent en bordure de lésion et créent un microenvironnement favorable à la régénération. Divers test fonctionnels in vivo et in vitro révèlent le rôle clef des HSPGs dans la cinétique de démyélinisation et de remyélinisation en régulant la mobilisation des cellules du lignage oligodendrocytaire et l’activation microgliale. / In the mammal’s nervous system, the ongoing production of new myelinating cells on has open news therapeutic perspectives for demyelinating diseases. An endogenous regeneration process involving the generation of oligodendrocytes can occur following demyelination. This process relies on the mobilization of an endogenous reservoir of progenitor cells located in the adult brain: The parenchymal oligodendrocyte precursors and the subventricular zone derived neural progenitors. However, these endogenous repair attempts do not permit an efficient functional recovery. These failures are mainly due to mobilization, differentiation or to the generation of a hostile environment for the repair process. Our work is focusing on the identification of factors regulating those events. Our data show the reexpression of CNTF and overexpression of heparan sulphate proteoglycans (HSPGs) following a focal demyelination of the corpus callosum in adult mice. These environmental changes favor myelin repair. We show a direct impact of the post-lesional expression of CNTF on the mobilization of both cellular sources. Using various in vitro assays, we showed that CNTF is acting on the two cellular sources as a chemoattractant factor. Our data also show that sulfation modifications of HSPGs performed by the deacetylase-N-sulfotransferase 1 (Ndst1) on oligodendrocyte lineage cells occurred around the lesion and created a permissive microenvironment for the regenerative process. Various in vitro and in vivo functional assays demonstrated the key role of HSPGs in demyelination and remyelination dynamic by controlling mobilization of the oligodendrocyte lineage cells and microglial activation.