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Common in vitro models used to study the mechanisms regulating myelination rely on co-cultures of oligodendrocyte precursor cells (OPCs) and neurons. In such models, myelination occurs in an environment that does not fully reflect cell-cell interactions and environmental cues present in vivo. To avoid these limitations while specifically manipulating oligodendroglial cells, we developed a reliable ex vivo model of myelination by seeding OPCs on cerebellar slices, deprived of their endogenous oligodendrocytes. We showed that exogenous OPCs seeded on unmyelinated cerebella, efficiently differentiate and form compact myelin. Spectral confocal reflectance microscopy and electron microscopy analysis revealed that the density of compacted myelin sheaths highly increases all along the culture. Importantly, we defined the appropriate culture time frame to study OPC differentiation and myelination, using accurate quantification resources we generated. Thus, this model is a powerful tool to study the cellular and molecular mechanisms of OPC differentiation and myelination. Moreover, it is suitable for the development and validation of new therapies for myelin-related disorders such as multiple sclerosis and psychiatric diseases.

White matter disorders of the CNS such as MS, lead to failure of nerve conduction and long-lasting neurological disabilities affecting a variety of sensory and motor systems including vision. While most disease-modifying therapies target the immune and inflammatory response, the promotion of remyelination has become a new therapeutic avenue, to prevent neuronal degeneration and promote recovery. Most of these strategies are developed in short-lived rodent models of demyelination, which spontaneously repair and do not reflect the size, organization, and biology of the human CNS. Thus, well-defined non-human primate models are required to efficiently advance therapeutic approaches for patients. Here, we followed the consequence of long-term toxin-induced demyelination of the macaque optic nerve on remyelination and axon preservation, as well as its impact on visual functions. Findings from oculo-motor behavior, ophthalmic examination, electrophysiology, and retinal imaging indicate visual impairment involving the optic nerve and retina. These visual dysfunctions fully correlated at the anatomical level, with sustained optic nerve demyelination, axonal degeneration, and alterations of the inner retinal layers. This non-human primate model of chronic optic nerve demyelination associated with axonal degeneration and visual dysfunction, recapitulates several key features of MS lesions and should be instrumental in providing the missing link to translate emerging repair pro-myelinating/neuroprotective therapies to the clinic for myelin disorders such as MS.Significance StatementPromotion of remyelination has become a new therapeutic avenue, to prevent neuronal degeneration and promote recovery in white matter diseases such as MS. To date most of these strategies are developed in short-lived rodent models of demyelination, which spontaneously repair. Well-defined non-human primate models closer to man would allow to efficiently advance therapeutic approaches. Here we present a non-human primate model of optic nerve demyelination that recapitulates several features of MS lesions. The model leads to failed remyelination, associated with progressive axonal degeneration and visual dysfunction, thus providing the missing link to translate emerging pre-clinical therapies to the clinic for myelin disorders such as MS.

The presence of putative stem/progenitor cells has been suggested in adult peripheral nervous system (PNS) tissue, including the dorsal root ganglion (DRG). To date, their identification and fate in pathophysiological conditions have not been addressed. Combining multiple in vitro and in vivo approaches, we identified the presence of stem cells in the adult DRG satellite glial population, and progenitors were present in the DRGs and sciatic nerve. Cell-specific transgenic mouse lines highlighted the proliferative potential of DRG stem cells and progenitors in vitro. DRG stem cells had gliogenic and neurogenic potentials, whereas progenitors were essentially gliogenic. Lineage tracing showed that, under physiological conditions, adult DRG stem cells maintained DRG homeostasis by supplying satellite glia. Under pathological conditions, adult DRG stem cells replaced DRG neurons lost to injury in addition of renewing the satellite glial pool. These novel findings open new avenues for development of therapeutic strategies targeting DRG stem cells for PNS disorders.

International audience; Slit1 is a secreted axon guidance molecule, also involved in adult neurogenesis. In physiological conditions, Slit1 loss promotes ectopic dispersal of SVZ-derived neural precursors (SVZ-NPCs) into periventricular structures such as the corpus callosum. Demyelination of the corpus callosum triggers SVZ-NPC migration to ectopic locations and their recruitment by the lesion, suggesting a possible role for Slit1 in SVZ-NPCs ectopic dispersal regulation in pathological conditions. Here, we have investigated the function of Slit1 protein in the recruitment of SVZ-NPCs after CNS demyelination. We find that the dynamics of oligodendrogenesis and temporal profile of developmental myelination in Slit1 -/- mice are similar to Slit1 +/- controls. SVZ micro-dissection and RT-PCR from wild-type mice, show that Slits and Robos are physiologically regulated at the transcriptional level in response to corpus callosum demyelination suggesting their role in the process of SVZ-NPC ectopic migration in demyelinating conditions. Moreover, we find that the number of SVZ-NPCs recruited by the lesion increases in Sli1-/- mice compared to Slit1 +/- mice, leading to higher numbers of Olig2+ cells within the lesion. Time-lapse video-microscopy of immuno-purified NPCs shows that Slit1-deficient cells migrate faster and make more frequent directional changes than control NPCs, supporting a cell-autonomous mechanism of action of Slit1 in NPC migration. In conclusion, while Slit1 does not affect the normal developmental process of oligodendrogenesis and myelination, it regulates adult SVZ-NPC ectopic migration in response to demyelination, and consequently oligodendrocyte renewal within the lesion.

Background and ObjectivesTo test whether low concentrations of teriflunomide (TF) could promote remyelination, we investigate the effect of TF on oligodendrocyte in culture and on remyelination in vivo in 2 demyelinating models.MethodsThe effect of TF on oligodendrocyte precursor cell (OPC) proliferation and differentiation was assessed in vitro in glial cultures derived from neonatal mice and confirmed on fluorescence-activated cell sorting–sorted adult OPCs. The levels of the 8,9-unsaturated sterols lanosterol and zymosterol were quantified in TF- and sham-treated cultures. In vivo, TF was administered orally, and remyelination was assessed both in myelin basic protein–GFP-nitroreductase (Mbp:GFP-NTR) transgenic Xenopus laevis demyelinated by metronidazole and in adult mice demyelinated by lysolecithin.ResultsIn cultures, low concentrations of TF down to 10 nM decreased OPC proliferation and increased their differentiation, an effect that was also detected on adult OPCs. Oligodendrocyte differentiation induced by TF was abrogated by the oxidosqualene cyclase inhibitor Ro 48-8071 and was mediated by the accumulation of zymosterol. In the demyelinated tadpole, TF enhanced the regeneration of mature oligodendrocytes up to 2.5-fold. In the mouse demyelinated spinal cord, TF promoted the differentiation of newly generated oligodendrocytes by a factor of 1.7-fold and significantly increased remyelination.DiscussionTF enhances zymosterol accumulation in oligodendrocytes and CNS myelin repair, a beneficial off-target effect that should be investigated in patients with multiple sclerosis.

Schwann cells (SC) enter the central nervous system (CNS) in pathophysiological conditions. However, how SC invade the CNS to remyelinate central axons remains undetermined. We studied SC migratory behavior ex vivo and in vivo after exogenous transplantation in the demyelinated spinal cord. The data highlight for the first time that SC migrate preferentially along blood vessels in perivascular extracellular matrix (ECM), avoiding CNS myelin. We demonstrate in vitro and in vivo that this migration route occurs by virtue of a dual mode of action of Eph/ephrin signaling. Indeed, EphrinB3, enriched in myelin, interacts with SC Eph receptors, to drive SC away from CNS myelin, and triggers their preferential adhesion to ECM components, such as fibronectin via integrinβ1 interactions. This complex interplay enhances SC migration along the blood vessel network and together with lesion-induced vascular remodeling facilitates their timely invasion of the lesion site. These novel findings elucidate the mechanism by which SC invade and contribute to spinal cord repair. Electronic supplementary material The online version of this article (10.1007/s00401-019-02011-1) contains supplementary material, which is available to authorized users.

Induced pluripotent stem cell–derived (iPS-derived) neural precursor cells may represent the ideal autologous cell source for cell-based therapy to promote remyelination and neuroprotection in myelin diseases. So far, the therapeutic potential of reprogrammed cells has been evaluated in neonatal demyelinating models. However, the repair efficacy and safety of these cells has not been well addressed in the demyelinated adult CNS, which has decreased cell plasticity and scarring. Moreover, it is not clear if these induced pluripotent–derived cells have the same reparative capacity as physiologically committed CNS-derived precursors. Here, we performed a side-by-side comparison of CNS-derived and skin-derived neural precursors in culture and following engraftment in murine models of adult spinal cord demyelination. Grafted induced neural precursors exhibited a high capacity for survival, safe integration, migration, and timely differentiation into mature bona fide oligodendrocytes. Moreover, grafted skin–derived neural precursors generated compact myelin around host axons and restored nodes of Ranvier and conduction velocity as efficiently as CNS-derived precursors while outcompeting endogenous cells. Together, these results provide important insights into the biology of reprogrammed cells in adult demyelinating conditions and support use of these cells for regenerative biomedicine of myelin diseases that affect the adult CNS.

Oligodendrocyte development is a critical process timely and spatially regulated to ensure proper myelination of the central nervous system. HMG-box transcription factors are key regulators of oligodendrocyte lineage progression. Among these factors, Sox17 was previously identified as a positive regulator of oligodendrocyte development. However, the role of Sox17 in oligodendroglial cell lineage progression and differentiation is still poorly understood. To define the functional role of Sox17, we generated new transgenic mouse models with inducible overexpression of Sox17, specifically in oligodendroglial cells. Here, we report that gain of Sox17 function has no effect on oligodendrocyte progenitor cells (OPCs) specification. During early postnatal development, Sox17 overexpression increases the pool of OPCs at the expense of differentiated oligodendrocytes. However, the oligodendroglial cell population, OPC proliferation and apoptosis remained unchanged in Sox17 transgenic mice. RNA sequencing, quantitative RT-PCR and immunohistochemical analysis showed that Sox17 represses the expression of the major myelin genes, resulting in a severe CNS hypomyelination. Overall, our data highlight an unexpected role for Sox17 as a negative regulator of OPC differentiation and myelination, suggesting stage specific functions for this factor during oligodendroglial cell lineage progression.

Remyelination of CNS axons by Schwann cells (SCs) is not efficient, in part due to the poor migration of SCs into the adult CNS. Although it is known that migrating SCs avoid white matter tracts, the molecular mechanisms underlying this exclusion have never been elucidated. We now demonstrate that myelin-associated glycoprotein (MAG), a well known inhibitor of neurite outgrowth, inhibits rat SC migration and induces their death via γ-secretase-dependent regulated intramembrane proteolysis of the p75 neurotrophin receptor (also known as p75 cleavage). Blocking p75 cleavage using inhibitor X (Inh X), a compound that inhibits γ-secretase activity before exposing to MAG or CNS myelin improves SC migration and survival in vitro Furthermore, mouse SCs pretreated with Inh X migrate extensively in the demyelinated mouse spinal cord and remyelinate axons. These results suggest a novel role for MAG/myelin in poor SC-myelin interaction and identify p75 cleavage as a mechanism that can be therapeutically targeted to enhance SC-mediated axon remyelination in the adult CNS.SIGNIFICANCE STATEMENT Numerous studies have used Schwann cells, the myelin-making cells of the peripheral nervous system to remyelinate adult CNS axons. Indeed, these transplanted cells successfully remyelinate axons, but unfortunately they do not migrate far and so remyelinate only a few axons in the vicinity of the transplant site. It is believed that if Schwann cells could be induced to migrate further and survive better, they may represent a valid therapy for remyelination. We show that myelin-associated glycoprotein or CNS myelin, in general, inhibit rodent Schwann cell migration and induce their death via cleavage of the neurotrophin receptor p75. Blockade of p75 cleavage using a specific inhibitor significantly improves migration and survival of the transplanted Schwann cells in vivo.

Rapid and efficient protocols to generate oligodendrocytes (OL) from human induced pluripotent stem cells (iPSC) are currently lacking, but may be a key technology to understand the biology of myelin diseases and to develop treatments for such disorders. Here, we demonstrate that the induction of three transcription factors (SOX10, OLIG2, NKX6.2) in iPSC-derived neural progenitor cells is sufficient to rapidly generate O4+ OL with an efficiency of up to 70% in 28 d and a global gene-expression profile comparable to primary human OL. We further demonstrate that iPSC-derived OL disperse and myelinate the CNS of Mbpshi/shiRag-/- mice during development and after demyelination, are suitable for in vitro myelination assays, disease modeling, and screening of pharmacological compounds potentially promoting oligodendroglial differentiation. Thus, the strategy presented here to generate OL from iPSC may facilitate the studying of human myelin diseases and the development of high-throughput screening platforms for drug discovery.

International audience; Oligodendrocyte and myelin deficits have been reported in mental/psychiatric diseases. The p21-activated kinase 3 (PAK3), a serine/threonine kinase, whose activity is stimulated by the binding of active Rac and Cdc42 GTPases is affected in these pathologies. Indeed, many mutations of Pak3 gene have been described in non-syndromic intellectual disability diseases. Pak3 is expressed mainly in the brain where its role has been investigated in neurons but not in glial cells. Here, we showed that PAK3 is highly expressed in oligodendrocyte precursors (OPCs) and its expression decreases in mature oligodendrocytes. In the developing white matter of the Pak3 knockout mice, we found defects of oligodendrocyte differentiation in the corpus callosum and to a lesser extent in the anterior commissure, which were compensated at the adult stage. In vitro experiments in OPC cultures, derived from Pak3 knockout and wild type brains, support a developmental and cell-autonomous role for PAK3 in regulating OPC differentiation into mature oligodendrocytes. Moreover, we did not detect any obvious alterations of the proliferation or migration of Pak3 null OPCs compared to wild type. Overall, our data highlight PAK3 as a new regulator of OPC differentiation.

The factors that determine whether remyelination fails or succeeds in multiple sclerosis remain unknown. By grafting lymphocytes from patients into demyelinated lesions in mice, El Behi, Sanson et al. show that lymphocytes differ in their ability to induce remyelination. Unravelling the basis of this heterogeneity reveals prerequisites for efficient myelin repair., One major challenge in multiple sclerosis is to understand the cellular and molecular mechanisms leading to disease severity progression. The recently demonstrated correlation between disease severity and remyelination emphasizes the importance of identifying factors leading to a favourable outcome. Why remyelination fails or succeeds in multiple sclerosis patients remains largely unknown, mainly because remyelination has never been studied within a humanized pathological context that would recapitulate major events in plaque formation such as infiltration of inflammatory cells. Therefore, we developed a new paradigm by grafting healthy donor or multiple sclerosis patient lymphocytes in the demyelinated lesion of nude mice spinal cord. We show that lymphocytes play a major role in remyelination whose efficacy is significantly decreased in mice grafted with multiple sclerosis lymphocytes compared to those grafted with healthy donors lymphocytes. Mechanistically, we demonstrated in vitro that lymphocyte-derived mediators influenced differentiation of oligodendrocyte precursor cells through a crosstalk with microglial cells. Among mice grafted with lymphocytes from different patients, we observed diverse remyelination patterns reproducing for the first time the heterogeneity observed in multiple sclerosis patients. Comparing lymphocyte secretory profile from patients exhibiting high and low remyelination ability, we identified novel molecules involved in oligodendrocyte precursor cell differentiation and validated CCL19 as a target to improve remyelination. Specifically, exogenous CCL19 abolished oligodendrocyte precursor cell differentiation observed in patients with high remyelination pattern. Multiple sclerosis lymphocytes exhibit intrinsic capacities to coordinate myelin repair and further investigation on patients with high remyelination capacities will provide new pro-regenerative strategies.

Pelizaeus-Merzbacher disease (PMD) results from an X-linked misexpression of proteolipid protein 1 (PLP1). This leukodystrophy causes severe hypomyelination with progressive inflammation, leading to neurological dysfunctions and shortened life expectancy. While no cure exists for PMD, experimental cell-based therapy in the dysmyelinated shiverer model suggested that human oligodendrocyte progenitor cells (hOPCs) or human neural precursor cells (hNPCs) are promising candidates to treat myelinopathies. However, the fate and restorative advantages of human NPCs/OPCs in a relevant model of PMD has not yet been addressed. Using a model of Plp1 overexpression, resulting in demyelination with progressive inflammation, we compared side-by-side the therapeutic benefits of intracerebrally grafted hNPCs and hOPCs. Our findings reveal equal integration of the donor cells within presumptive white matter tracks. While the onset of exogenous remyelination was earlier in hOPCs-grafted mice than in hNPC-grafted mice, extended lifespan occurred only in hNPCs-grafted animals. This improved survival was correlated with reduced neuroinflammation (microglial and astrocytosis loads) and microglia polarization toward M2-like phenotype followed by remyelination. Thus modulation of neuroinflammation combined with myelin restoration is crucial to prevent PMD pathology progression and ensure successful rescue of PMD mice. These findings should help to design novel therapeutic strategies combining immunomodulation and stem/progenitor cell-based therapy for disorders associating hypomyelination with inflammation as observed in PMD.