Antagonistic actions of PAK1 and NF2/Merlin drive myelin membrane expansion in oligodendrocytes.

In the central nervous system, the formation of myelin by oligodendrocytes (OLs) relies on the switch from the polymerization of the actin cytoskeleton to its depolymerization. The molecular mechanisms that trigger this switch have yet to be elucidated. Here, we identified P21-activated kinase 1 (PAK1) as a major regulator of actin depolymerization in OLs. Our results demonstrate that PAK1 accumulates in OLs in a kinase-inhibited form, triggering actin disassembly and, consequently, myelin membrane expansion. Remarkably, proteomic analysis of PAK1 binding partners enabled the identification of NF2/Merlin as its endogenous inhibitor. Our findings indicate that Nf2 knockdown in OLs results in PAK1 activation, actin polymerization, and a reduction in OL myelin membrane expansion. This effect is rescued by treatment with a PAK1 inhibitor. We also provide evidence that the specific Pak1 loss-of-function in oligodendroglia stimulates the thickening of myelin sheaths in vivo. Overall, our data indicate that the antagonistic actions of PAK1 and NF2/Merlin on the actin cytoskeleton of the OLs are critical for proper myelin formation. These findings have broad mechanistic and therapeutic implications in demyelinating diseases and neurodevelopmental disorders.

Satellite glia of the adult dorsal root ganglia harbor stem cells that yield glia under physiological conditions and neurons in response to injury.

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.

Failed remyelination of the nonhuman primate optic nerve leads to axon degeneration, retinal damages, and visual dysfunction.

White matter disorders of the central nervous system (CNS), such as multiple sclerosis (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 have been 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 nonhuman 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 oculomotor 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 nonhuman 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 promyelinating/neuroprotective therapies to the clinic for myelin disorders, such as MS.

Teriflunomide Promotes Oligodendroglial 8,9-Unsaturated Sterol Accumulation and CNS Remyelination.

BACKGROUND AND OBJECTIVES: To 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. METHODS: The 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. RESULTS: In 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. DISCUSSION: TF enhances zymosterol accumulation in oligodendrocytes and CNS myelin repair, a beneficial off-target effect that should be investigated in patients with multiple sclerosis.

Co-culture of exogenous oligodendrocytes with unmyelinated cerebella: Revisiting ex vivo models and new tools to study myelination.

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.

Blood vessels guide Schwann cell migration in the adult demyelinated CNS through Eph/ephrin signaling.

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 integrinß1 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.

SOX17 transcription factor negatively regulates oligodendrocyte precursor cell differentiation.

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.

Myelin-Associated Glycoprotein Inhibits Schwann Cell Migration and Induces Their Death.

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.

Adaptive human immunity drives remyelination in a mouse model of demyelination.

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.

Rapid and efficient generation of oligodendrocytes from human induced pluripotent stem cells using transcription factors.

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.