A key problem in multiple sclerosis (MS) is the diminished capacity for myelin repair. Although oligodendrocyte (OL) precursors can be seen at the lesion site, their ability to differentiate appears inhibited. MicroRNAs are key regulators of OL differentiation, and have been observed to be misregulated in MS lesions compared to healthy white matter. Thus, aberrant microRNA expression in MS lesions may disrupt the ability of incoming oligodendrocyte progenitor cells (OPC s) to differentiate. Specifically, a microRNA known as miR - 145 is downregulated as OPCs progress to OLs, but is found at unusually high levels in MS lesions. In this study, we investigated how misregulation of miR - 145 affects OL differentiation in vitro. Bioinformatic analysis revealed that putative targets of miR - 145 are significantly enriched for factors which promote actin cytoskeleton organization and myelination. An immortalized OL cell line was transduced with an inducible lentivirus to create stable lines that overexpress miR - 145. These stable lines were characterized while proliferating, early in differentiation and late in differentiation. Immunofluorescence was used to quantify changes in proliferation rate, apoptosis, branching ability and myelin gene expression. qPCR arrays were used to quantify changes in microRNA target expression levels between induced and uninduced cells. Two stable lines were created: ON - 145 - 1 and ON - 145 - 2, which upon induction, over - express miR - 145 ~33 - fold and ~11 - fold, respectively. When proliferating, no significant morphological differences nor target expression differences could be detected between induced and uninduced cells. Proliferation was significantly decreased in ON - 145 - 1 induced cells, but not in ON - 145 - 2. No changes in apoptosis frequency were detected. In contrast, during early and late differentiation, both induced cell lines showed significant morphological defects characterized by a reduction in both iii primary and secondary branching. Further, significant differences in branching ability were observed between induced cells of ON - 145 - 1 and ON - 145 - 2, suggesting a dose - dependent response to miR - 145 overexpression. Expression of MAG, a myelin marker, was also significantly lowered in induced cells of both cell lines. Finally, we found that multiple miR - 145 targets involved in promoting cytoskeletal organization and myelination were significantly decreased both early and late in differentiation. These results suggest that overexpression of miR - 145 during OL differentiation may disrupt actin organization and myelin gene expression required for successful process extension and subsequent myelinating ability. Thus, the increase in miR - 145 in MS lesions may be a significant contributing factor to the loss of myelin repair in MS lesions.

While the classical function of myelin is to facilitate saltatory conduction, this membrane and the myelin-making oligodendrocytes (OLGs) are now recognized as regulators of plasticity and remodeling in the central nervous system (CNS). Thus, OLG maturation and myelination are highly vulnerable processes along CNS development. We previously showed that rat brain myelination is altered by perinatal exposure to buprenorphine, an opioid analogue in clinical trials for the treatment of pregnant opioid addicts. We now found that the in vivo effects on myelination could result from direct alteration in the balance between μ-opioid receptor (MOR) and nociceptin/orphanin FQ receptor (NOPR) activities in the OLGs. Furthermore, we found that myelination could also be affected by the FDA-approved methadone. A delicate balance between MOR and NOPR signaling may play a crucial role timing OLG maturation and myelin formation and exogenous opioids may disrupt this interplay, altering the developmental pattern of brain myelination.

La démyélinisation et la mort oligodendrocytaire sont bien connues dans la sclérose en plaques (SEP). Au cours de ces dernières années, plusieurs études ont également décrit ce type de lésion après un traumatisme crânien (TC), participant à l’aggravation des lésions de la substance blanche, responsables des dysfonctionnements cognitifs et moteurs. Malgré de nombreux efforts, aucune thérapie efficace n’est disponible à ce jour pour traiter les lésions de la substance blanche. Dans ce contexte, une stratégie thérapeutique prometteuse serait de freiner la neuro-inflammation et la démyélinisation, en plus de promouvoir la maturation des oligodendrocytes afin de favoriser la remyélinisation des axones et de limiter ainsi leur dégénérescence. Notre choix de stratégie porte sur la stimulation des processus de réparation endogène via la protéine neuroprotectrice et neurotrophique sAPPα, forme soluble de la protéine βAPP libérée par l’action des α-sécrétases (ADAM10). Dans ce contexte, l’objectif de mes travaux de thèse était d’étudier l’intérêt thérapeutique de l’étazolate, un activateur desα-sécrétases, sur les conséquences biochimiques, histologiques et fonctionnelles, dans différents modèles de TC et de SEP in vivo chez la souris, et ex vivo sur des tranches organotypiques de cervelet. Les résultats obtenus sur le modèle de TC par percussion mécanique chez la souris ont montré pour la première fois le potentiel anti-inflammatoire de l’étazolate, associé à la restauration du taux de la sAPPα. De plus, l’étazolate s’est également opposé aux troubles fonctionnels post-TC tels que l’hyperactivité locomotrice et le déficit cognitif à court et à long terme. Par la suite, j’ai développé un nouveau modèle ex vivo de TC par percussion mécanique sur des tranches organotypiques de cervelet. Nous avons montré pour la première fois que l’étazolate était neuroprotecteur dans le tissu cérébelleux, et qu’il s’opposait à la démyélinisation post-traumatique. Par ailleurs, les effets bénéfiques de l’étazolate sur les gaines de myéline ont été reproduits dans un modèle ex vivo de démyélinisation induite par la lysolécithine, modèle ex vivo de SEP. De façon intéressante nous avons montré que l’étazolate exerçait un effet remyélinisant en stimulant la différenciation des oligodendrocytes. Cet effet a été reproduit in vitro dans des cultures primaires mixtes de cellules gliales issues de souris PLP-eGFP, où la maturation morphologique des oligodendrocytes a été favorisée en présence d’étazolate. L’ensemble des effets bénéfiques exercés par l’étazolate a été inhibé en présence d’un inhibiteur pharmacologique spécifique d’ADAM10, le GI254023X, suggérant que l’effet remyélinisant de l’étazolate dépend, au moins en partie, d’ADAM10. Par la suite, l’effet remyélinisant de l’étazolate a été étudié dans un modèle in vivo de démyélinisation chronique induite par la cuprizone. Dans ce modèle, l’étazolate a été capable de promouvoir la remyélinisation en stimulant la différenciation des oligodendrocytes, confirmant nos résultats in vitro et ex vivo. L’ensemble de mon travail permet de considérer le potentiel thérapeutique de l’étazolate, en visant l’ADAM10 comme nouvelle cible thérapeutique neuroprotectrice et remyélinisante. Cela aura pour intérêt de limiter la neuro-inflammation, la démyélinisation, ainsi que de promouvoir la différenciation des oligodendrocytes et la remyélinisation, afin de favoriser la récupération fonctionnelle suite aux lésions de la substance blanche survenant après un TC ou la SEP chez l’homme. / Demyelination and oligodendrocyte cell death are well established in multiple sclerosis (MS) and are increasingly described after traumatic brain injury (TBI), participating in the aggravation of white matter injury responsible of motor and cognitive deficits. Despite many efforts in clinical research, no efficient therapy against white matter injury progression is available nowadays. Thus, promoting remyelination and counteracting neuroinflammation and demyelination are major therapeutic strategies in order to restore white matter integrity. Here, we studied the stimulation of endogenous repair mechanisms through the neuroprotective and neurotrophic protein sAPPα, the soluble form of βAPP protein released by the α-secretases (ADAM10). In this context, the aim of this work was to evaluate the therapeutic potential of etazolate, an α-secretase activator on short- and long-term biochemical, histological and functional outcome in different mouse models of TBI and MS in vivo, and ex vivo on organotypic cerebellar slices. The results obtained from the TBI mouse model by mechanical percussion showed for the first time the anti-inflammatory effect of etazolate associated to a restoration of sAPPα levels. The same treatment was able to attenuate functional deficits (hyperactivity, cognitive deficit). We also developed a new ex vivo model of TBI by mechanical percussion on organotypic cerebellar slices. We confirmed the neuroprotective effect of etazolate on cerebellar tissue reducing the lesion size. Interestingly, etazolate treatment attenuated post-traumatic ex vivo demyelination. Moreover, the beneficial effect of etazolate on myelin sheaths have been well reproduced after lysolecithin-induced demyelination, an ex vivo model of MS. Interestingly, etazolate was able to enhance remyelination promoting oligodendrocyte differentiation. This effect has been reproduced in the primary mixed glial culture from PLP-eGFP mice, enhancing oligodendrocyte morphological maturation. However, etazolate failed to promote its beneficial effects in the presence of GI254023X, a specific ADAM10 (α-secretase) inhibitor, suggesting that the mechanism of action of etazolate is partly through the activation of ADAM10. Furthermore, etazolate reproduced in vivo the oligodendrocyte differentiation, accompanied by an increase of the myelinated axons, observed by electron microscopy in a mouse model of cuprizone-induced chronic demyelination. Taken together, the findings of this work provide a first evidence for the therapeutic potential of etazolate, with ADAM10 as new therapeutic target in white matter repair. The interest of this approach is to attenuate neuroinflammation and demyelination and to enhance oligodendrocyte differentiation and thus remyelination, in order to promote functional recovery following white matter lesions arising after TBI or MS.