La sclérose en plaques (SP) est une maladie neuro-inflammatoire caractérisée par l’invasion de cellules immunitaires périphériques dans le système nerveux central (SNC), entraînant une perte de myéline à des endroits bien délimités appelés « plaques » ou lésions. Les processus neuroinflammatoires sont associés au dommage des neurones et oligodendrocytes (OLs) en SP. Les mécanismes sous-tendant cette dégradation des OLs par les cellules immunitaires en SP sont toutefois encore mal compris. Les lymphocytes T CD4 activés, notamment les sous-types proinflammatoires Th1 et Th17, jouent un rôle clé dans la pathobiologie de la SP et de son modèle murin l’encéphalite auto-immune expérimentale (EAE). Nous avons donc choisi d’investiguer leur contribution à l’endommagement des OLs en neuroinflammation. Pour ce faire, nous avons premièrement caractérisé les interactions entre les lymphocytes Th17 et les OLs matures in vivo à l’aide de l’imagerie intravitale chez la souris EAE (microscopie deux photons) et in vitro en utilisant des cultures primaires humaines. Ceci nous a permis de mettre en évidence que les lymphocytes pro-inflammatoires Th17 adhèrent de façon prolongée aux OLs et leur causent plus de dommage que les lymphocytes anti-inflammatoires Th2. Après avoir établi que le contact avec les lymphocytes Th17 entraîne tout d’abord la perte des prolongements cellulaires puis la mort des OLs, nous avons identifié deux mécanismes à l’origine de ces dommages. En effet, tandis que la sécrétion de glutamate par les lymphocytes Th17 à proximité des OLs entraîne une perte des prolongements cellulaires de ces derniers et une diminution de leur capacité à myéliniser, la sécrétion de granzyme B mène à la mort des OLs. Dans le but de comprendre comment prévenir les dommages causés par les lymphocytes Th17 aux OLs en SP, nous avons par la suite étudié les mécanismes sous-tendant le contact entre les deux types cellulaires. Comme nous avons confirmé que les OLs matures n’expriment pas le MHC II au niveau protéique, nous avons caractérisé l’expression par les OLs de molécules d’adhérence cellulaire (CAMs) qui seraient susceptibles de sous-tendre l’adhérence des lymphocytes Th17. Nous avons découvert que cette interaction est notamment médiée par ALCAM, et que bloquer cette molécule permet de diminuer le dommage aux OLs médié par les Th17 in vitro. A l’inverse, l’expression et/ou la sécrétion d’ICAM-1 par les OLs semble avoir un effet protecteur face aux lymphocytes Th17. En résumé, nous avons distingué de nouveaux mécanismes impliqués dans le dommage aux OLs en neuroinflammation et identifié de nouvelles cibles thérapeutiques prometteuses pour la protection des OLs en SP. / Multiple Sclerosis (MS) is a neuroinflammatory disease characterized by infiltration of immune cells into the central nervous system (CNS), demyelination in multifocal areas called “plaques” or lesions, and damage to neurons and oligodendrocytes (OLs). The mechanisms underlying immune-mediated injury to OLs in MS remains only partially understood. Activated CD4 T cells, in particular pro-inflammatory subsets Th1 and Th17, play an important role in the pathobiology of MS and its animal model experimental autoimmune encephalitis (EAE). We set out to investigate their contribution to immune-mediated oligodendrocytic damage in neuroinflammation. We first characterized the interactions between Th17 cells and mature OLs in vivo using live imaging of EAE mice (two photon microscopy) and in vitro using human primary cell cultures. We found that pro-inflammatory Th17 cells form prolonged contacts with OLs and cause greater harm compared to anti-inflammatory Th2 cells. After demonstrating that contact with Th17 cells leads first to destruction of cell processes and then death of OLs, we identified two mechanisms underlying these deleterious impacts. Indeed, while secretion of glutamate by Th17 cells in contact with OLs is associated with damage to OLs cell processes and impairment of their myelinating capacity, secretion of granzyme B leads to OLs death. To better understand how to prevent Th17-mediated OLs injury in MS, we next studied mechanisms involved in the interaction between these two cell types. As we confirmed that mature OLs do not express MHC II at the protein level, we characterized expression of cell adhesion molecules (CAMs) by OLs that could mediate Th17 cell adhesion. We discovered that ALCAM contributes to OLs and Th17 cells interactions, and that blocking this olecule reduces Th17-mediated OL damage in vitro. Inversely, ICAM-1 expression and/or secretion by OLs seems to have a protective effect in neuroinflammatory conditions. In summary, we have uncovered new mechanisms implicated in OLs njury in neuroinflammation and have identified potential novel therapeutic targets for neuroprotection in MS.

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Ce travail de thèse s'inscrit dans le cadre d'une collaboration avec le groupe Guerbet. Il visait à caractériser la lésion qui fait suite à un accident vasculaire cérébral (AVC) ischémique en imagerie par résonance magnétique (IRM) grâce à des agents de contraste novateurs. Guerbet et leurs collaborateurs ont développés des USPIO ciblés (ultrasmall superparamagnetic iron oxide), particules de fer couplées à des peptides reconnaissant spécifiquement un biomarqueur. Dans un modèle d’ischémie cérébrale avec reperfusion réalisé chez la souris, nous avons recherché la capacité de ces agents à caractériser la lésion d’une part en terme de type de mort cellulaire par le ciblage de la phosphatidylsérine (PS), marqueur cellulaire externalisé au cours de l’apoptose, et d’autre part en terme de déficit vasculaire par le ciblage de VCAM-1, molécule d’adhésion impliquée dans le processus inflammatoire. En ce qui concerne l’apoptose, nous avons tout d’abord montré par immunohistochimie l’expression de caspase-3 active, marqueur apoptotique, dès 6 heures et jusqu’à 72 heures après l’ischémie. Cependant, en IRM, l’utilisation d’USPIO ciblant la PS (le P03234 et le P03675) n’a pas permis la détection du phénomène apoptotique. Actuellement d’autres agents de contraste de ce type sont en cours de développement chez Guerbet. En ce qui concerne l’inflammation vasculaire, l’étude de l’expression de VCAM-1 par immunohistochimie a montré l’apparition d’un marquage dès 6 heures après l’ischémie avec un maximum à 24 heures. L’utilisation d’un USPIO-VCAM-1 (le P03011) a permis de mettre en évidence sur les images IRM des zones d’hypointensités dans la lésion, ce qui correspond à la présence de particules de fer. L'analyse histologique de ces cerveaux a montré une colocalisation de l’USPIO avec sa cible VCAM-1, ce qui établit la preuve de concept. Ces travaux ont permis mettre en évidence la capacité d’USPIO développés par Guerbet à cibler des marqueurs biologiques, notamment VCAM-1, à la suite d’une ischémie cérébrale. Ceci suggère que ce type d’agent de contraste pourrait être un bon outil clinique pour la caractérisation de la lésion ischémique chez les patients victimes d’AVC. / This work is a collaboration with Guerbet group. It aimed to characterize the lesion that follows an ischemic stroke with magnetic resonance imaging (MRI) by innovative contrast agents. Guerbet developped targeted USPIO (ultrasmall superparamagnetic iron oxide), which are iron particles coupled to peptides which specifically bind a biomarker. In a mouse model of cerebral ischemia-reperfusion, we studied the capacity of these agents to characterize the lesion on the one hand in terms of cellular death by targeting phosphatidylserin (PS), a cellular marker externalized during apoptosis, and on the other hand in terms of vascular deficit by targeting VCAM-1, an adhesion molecule implied in the inflammatory process. Concerning apoptosis, we showed by immunohistochemistry the expression of active caspase-3, an apoptotic marker, between 6 and 72 hours after ischemia. Nevertheless on MRI, the use of USPIO targeting PS (both P03234 and P03675) did not allow us to detect the apoptotic phenomenon. Currently, other PS-targeted contrast agents are developed by Guerbet. Concerning vascular inflammation, the study of VCAM-1 by immunohistochemistry showed an up-regulated expression 6 hours after ischaemia which reached a maximum at 24 hours. VCAM-1-USPIO (P03011) induced a decrease of the MRI signal appearing as hypointense foci in the lesion, which correspond to iron particles. The histological analysis of these brains showed a colocalisation of the USPIO with its target VCAM-1, which establishes the proof of concept. This work showed the capacity of USPIO developed by Guerbet to target biological markers, particularly VCAM-1, following cerebral ischemia. This suggests that this kind of contrast agent could be a good clinical tool to characterize the ischemic lesion in patients suffering from stroke.

Morphologic and metabolic change is a known property of the adult brain. A number of behavioural tasks alter local cerebral blood flow and glucose utilisation. The expression of the glucose transporter 1 (GLUT1), which allows the entry of glucose to the brain, also has been shown to change in response to long-lasting neuronal activation. However, little is known about the effect of acute neuronal activation on GLUT1 expression. Using immunohistochemistry and Western blot, we investigated cerebral GLUT1 expression and vasculature density in mice undergoing a 48-hour voluntary wheel running period. The results showed that the striatum was the main region where GLUT1 protein was up-regulated: There was a trend for GLUT1 expression and blood vessels density to be associated with the distance run during the experiment. These results indicate that short-term increased neuronal activation is associated with rapid changes in glucose transport and possibly vascular remodelling.

Morphologic and metabolic change is a known property of the adult brain. A number of behavioural tasks alter local cerebral blood flow and glucose utilisation. The expression of the glucose transporter 1 (GLUT1), which allows the entry of glucose to the brain, also has been shown to change in response to long-lasting neuronal activation. However, little is known about the effect of acute neuronal activation on GLUT1 expression. Using immunohistochemistry and Western blot, we investigated cerebral GLUT1 expression and vasculature density in mice undergoing a 48-hour voluntary wheel running period. The results showed that the striatum was the main region where GLUT1 protein was up-regulated: There was a trend for GLUT1 expression and blood vessels density to be associated with the distance run during the experiment. These results indicate that short-term increased neuronal activation is associated with rapid changes in glucose transport and possibly vascular remodelling.

Morphologic and metabolic change is a known property of the adult brain. A number of behavioural tasks alter local cerebral blood flow and glucose utilisation. The expression of the glucose transporter 1 (GLUT1), which allows the entry of glucose to the brain, also has been shown to change in response to long-lasting neuronal activation. However, little is known about the effect of acute neuronal activation on GLUT1 expression. Using immunohistochemistry and Western blot, we investigated cerebral GLUT1 expression and vasculature density in mice undergoing a 48-hour voluntary wheel running period. The results showed that the striatum was the main region where GLUT1 protein was up-regulated: There was a trend for GLUT1 expression and blood vessels density to be associated with the distance run during the experiment. These results indicate that short-term increased neuronal activation is associated with rapid changes in glucose transport and possibly vascular remodelling.

Polysialic acid is a unique carbohydrate that decorates the surface of the neural cell adhesion molecule. Polysialic acid is an onco-developmental antigen, expressed in tumours principally of neuroendocrine origin, notably neuroblastoma, strongly correlating with invasion and metastasis. Polysialylation is regulated by two polysialyltransferase enzymes, PST (ST8SiaIV) and STX (ST8SiaII), with STX dominant in cancer. Post-development polysialic acid expression is only found at low levels in the brain, thus this could be a novel target for cancer therapy. It is hypothesized that inhibition of polysialyltransferase could lead to control of tumour dissemination and metastasis. The aims of this thesis were to develop tools and in vitro assays to screen novel polysialyltransferase inhibitors. A panel of tumour cell lines were characterised in terms of growth parameters (using the MTT assay) and polysialic acid expression. This includes a pair of isogenic C6 rat glioma cells (C6-STX and C6-WT) and naturally polysialic acid expressing neuroblastoma cells (SH-SY5Y). Following this, an in vitro assay was validated to screen modulation of polysialic acid expression by removing pre-existing polysialic acid expression using endoneuraminidase N and evaluated the amount of re-expression of polysialic acid using immunocytochemistry. Then, a functional assay was developed and validated for invasion, the matrigel invasion assay. Cytidine monophosphate (tool compound) significantly reduced polysialic acid surface expression and invasion. A panel of six novel polysialyltransferase inhibitors was screened for cytotoxicity, polysialic acid surface expression and invasion. Of the potential polysialyltransferase inhibitors evaluated, ICT3176 and ICT3172 were identified from virtual screening of Maybridge library and were emerged as the most promising inhibitors, demonstrating significant (p < 0.05) reduction in cell-surface polysialic acid re-expression and invasion in polysialic acid expressing cells. Furthermore, the specificity of compounds for polysialyltransferase (α-2,8-sialyltransferase) over other members of the wider sialyltransferase family (α-2,3- and α-2,6-sialyltransferases) was confirmed using differential lectin staining. These results demonstrated that small molecule inhibitors as STX is possible and provides suitable in vitro cell based assays to discovery more potent derivatives.

Morphologic and metabolic change is a known property of the adult brain. A number of behavioural tasks alter local cerebral blood flow and glucose utilisation. The expression of the glucose transporter 1 (GLUT1), which allows the entry of glucose to the brain, also has been shown to change in response to long-lasting neuronal activation. However, little is known about the effect of acute neuronal activation on GLUT1 expression. Using immunohistochemistry and Western blot, we investigated cerebral GLUT1 expression and vasculature density in mice undergoing a 48-hour voluntary wheel running period. The results showed that the striatum was the main region where GLUT1 protein was up-regulated: There was a trend for GLUT1 expression and blood vessels density to be associated with the distance run during the experiment. These results indicate that short-term increased neuronal activation is associated with rapid changes in glucose transport and possibly vascular remodelling.

Polysialic acid is aunique carbohydrate that decorates the surface of the neural cell adhesion molecule. Polysialic acidis an onco-developmental antigen, expressed in tumours principally of neuroendocrine origin, notably neuroblastoma,strongly correlating with invasion and metastasis. Polysialylation is regulated by two polysialyltransferase enzymes, PST(ST8SiaIV)and STX(ST8SiaII),withSTX dominant in cancer. Post-development polysialic acid expression is only found at low levels in the brain, thus this could be a novel target for cancer therapy. It is hypothesized that inhibition of polysialyltransferasecould lead to control of tumour dissemination and metastasis.The aims of this thesis were to develop tools and in vitro assays to screen novel polysialyltransferaseinhibitors. A panel of tumour cell lines were characterised in terms of growth parameters (using the MTT assay) and polysialic acid expression. This includes a pair of isogenic C6 rat glioma cells (C6-STX and C6-WT) and naturally polysialic acid expressing neuroblastoma cells(SH-SY5Y). Following this, an in vitro assay was validated to screen modulation of polysialic acid expression by removing pre-existing polysialic acid expression using endoneuraminidase N and evaluated the amount of re-expression of polysialic acid using immunocytochemistry. Then, a functional assay was developed and validated for invasion, the matrigel invasion assay. Cytidine monophosphate (tool compound) significantly reduced polysialic acidsurface expression and invasion. A panel of six novel polysialyltransferase inhibitors was screened for cytotoxicity, polysialic acidsurface expression and invasion. Of the potential polysialyltransferase inhibitorsevaluated, ICT3176 and ICT3172 were identified from virtual screening of Maybridge library and were emerged as the most promising inhibitors, demonstrating significant (p<0.05)reduction in cell-surface polysialic acidre-expression and invasion in polysialic acid expressing cells.Furthermore, the specificity of compounds for polysialyltransferase (α-2,8-sialyltransferase) over othermembers of the wider sialyltransferase family (α-2,3-and α-2,6-sialyltransferases) was confirmed using differential lectin staining. These results demonstrated that small molecule inhibitors as STX is possible and provides suitable in vitrocell based assays to discovery more potent derivatives.