A retroviral link to vertebrate myelination through retrotransposon-RNA-mediated control of myelin gene expression.

Myelin, the insulating sheath that surrounds neuronal axons, is produced by oligodendrocytes in the central nervous system (CNS). This evolutionary innovation, which first appears in jawed vertebrates, enabled rapid transmission of nerve impulses, more complex brains, and greater morphological diversity. Here, we report that RNA-level expression of RNLTR12-int, a retrotransposon of retroviral origin, is essential for myelination. We show that RNLTR12-int-encoded RNA binds to the transcription factor SOX10 to regulate transcription of myelin basic protein (Mbp, the major constituent of myelin) in rodents. RNLTR12-int-like sequences (which we name RetroMyelin) are found in all jawed vertebrates, and we further demonstrate their function in regulating myelination in two different vertebrate classes (zebrafish and frogs). Our study therefore suggests that retroviral endogenization played a prominent role in the emergence of vertebrate myelin.

Mertk-expressing microglia influence oligodendrogenesis and myelin modelling in the CNS.

BACKGROUND: Microglia, an immune cell found exclusively within the CNS, initially develop from haematopoietic stem cell precursors in the yolk sac and colonise all regions of the CNS early in development. Microglia have been demonstrated to play an important role in the development of oligodendrocytes, the myelin producing cells in the CNS, as well as in myelination. Mertk is a receptor expressed on microglia that mediates immunoregulatory functions, including myelin efferocytosis. FINDINGS: Here we demonstrate an unexpected role for Mertk-expressing microglia in both oligodendrogenesis and myelination. The selective depletion of Mertk from microglia resulted in reduced oligodendrocyte production in early development and the generation of pathological myelin. During demyelination, mice deficient in microglial Mertk had thinner myelin and showed signs of impaired OPC differentiation. We established that Mertk signalling inhibition impairs oligodendrocyte repopulation in Xenopus tadpoles following demyelination. CONCLUSION: These data highlight the importance of microglia in myelination and are the first to identify Mertk as a regulator of oligodendrogenesis and myelin ultrastructure.

Monitoring recovery after CNS demyelination, a novel tool to de-risk pro-remyelinating strategies.

In multiple sclerosis, while remarkable progress has been accomplished to control the inflammatory component of the disease, repair of demyelinated lesions is still an unmet need. Despite encouraging results generated in experimental models, several candidates favouring or promoting remyelination have not reached the expected outcomes in clinical trials. One possible reason for these failures is that, in most cases, during preclinical testing, efficacy was evaluated on histology only, while functional recovery had not been assessed. We have generated a Xenopus laevis transgenic model Tg(mbp:GFP-NTR) of conditional demyelination in which spontaneous remyelination can be accelerated using candidate molecules. Xenopus laevis is a classic model for in vivo studies of myelination because tadpoles are translucent. We reasoned that demyelination should translate into loss of sensorimotor functions followed by behavioural recovery upon remyelination. To this end, we measured the swimming speed and distance travelled before and after demyelination and during the ongoing spontaneous remyelination and have developed a functional assay based on the visual avoidance of a virtual collision. Here we show that alteration of these functional and clinical performances correlated well with the level of demyelination and that histological remyelination, assayed by counting in vivo the number of myelinating oligodendrocytes in the optic nerve, translated in clinical-functional recovery. This method was further validated in tadpoles treated with pro-remyelinating agents (clemastine, siponimod) showing that increased remyelination in the optic nerve was associated with functional improvement. Our data illustrate the potential interest of correlating histopathological parameters and functional-clinical parameters to screen molecules promoting remyelination in a simple in vivo model of conditional demyelination.

Endogenous clues promoting remyelination in multiple sclerosis.

PURPOSE OF REVIEW: The introduction some 30 years ago of ß-interferon, followed by a panel of immunomodulators and immunosuppressants has led to a remarkable improvement in the management of multiple sclerosis (MS) patients. Despite these noticeable progresses, which lower the number of relapses and thereby ameliorate patients' quality of life, preventing long-term progression of disability is still an unmet need, highlighting the necessity to develop therapeutic strategies aimed at repairing demyelinated lesions and protecting axons from degeneration. The capacity of human brain to self-regenerate demyelinated lesion has opened a field of research aimed at fostering this endogenous potential. RECENT FINDINGS: The pioneer electron microscopic evidence by Périer and Grégoire [Périer O, Grégoire A. Electron microscopic features of multiple sclerosis lesions. Brain 1965; 88:937-952] suggesting the capacity of human brain to self-regenerate demyelinated lesion has opened a field of research aimed at fostering this endogenous potential. Here we review some recently identified mechanisms involved in the remyelination process, focusing on the role of electrical activity and the involvement of innate immune cells. We then provide an update on current strategies promoting endogenous myelin repair. SUMMARY: Identification of therapeutic targets for remyelination has opened an active therapeutic field in MS. Although still in early phase trials, with heterogenous efficacy, the door for myelin regeneration in MS is now opened.

Increased Remyelination and Proregenerative Microglia Under Siponimod Therapy in Mechanistic Models.

BACKGROUND AND OBJECTIVES: Siponimod is an oral, selective sphingosine-1-phosphate receptor-1/5 modulator approved for treatment of multiple sclerosis. METHODS: Mouse MRI was used to investigate remyelination in the cuprizone model. We then used a conditional demyelination Xenopus laevis model to assess the dose-response of siponimod on remyelination. In experimental autoimmune encephalomyelitis-optic neuritis (EAEON) in C57Bl/6J mice, we monitored the retinal thickness and the visual acuity using optical coherence tomography and optomotor response. Optic nerve inflammatory infiltrates, demyelination, and microglial and oligodendroglial differentiation were assessed by immunohistochemistry, quantitative real-time PCR, and bulk RNA sequencing. RESULTS: An increased remyelination was observed in the cuprizone model. Siponimod treatment of demyelinated tadpoles improved remyelination in comparison to control in a bell-shaped dose-response curve. Siponimod in the EAEON model attenuated the clinical score, reduced the retinal degeneration, and improved the visual function after prophylactic and therapeutic treatment, also in a bell-shaped manner. Inflammatory infiltrates and demyelination of the optic nerve were reduced, the latter even after therapeutic treatment, which also shifted microglial differentiation to a promyelinating phenotype. DISCUSSION: These results confirm the immunomodulatory effects of siponimod and suggest additional regenerative and promyelinating effects, which follow the dynamics of a bell-shaped curve with high being less efficient than low concentrations.

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.

Evolution of glial wrapping: A new hypothesis.

Animals are able to move and react in numerous ways to external stimuli. Thus, environmental stimuli need to be detected, information must be processed and finally an output decision must be transmitted to the musculature to get the animal moving. All these processes depend on the nervous system which comprises an intricate neuronal network and many glial cells. In the last decades, a neurono-centric view on nervous system function channeled most of the scientific interest toward the analysis of neurons and neuronal functions. Neurons appeared early in animal evolution and the main principles of neuronal function from synaptic transmission to propagation of action potentials are conserved during evolution. In contrast, not much is known on the evolution of glial cells that were initially considered merely as static support cells. Although it is now accepted that glial cells have an equally important contribution as their neuronal counterpart to nervous system function, their evolutionary origin is unknown. Did glial cells appear several times during evolution? What were the first roles glial cells had to fulfil in the nervous system? What triggered the formation of the amazing diversity of glial morphologies and functions? Is there a possible mechanism that might explain the appearance of complex structures such as myelin in vertebrates? Here, we postulate a common evolutionary origin of glia and depict a number of selective forces that might have paved the way from a simple supporting cell to a wrapping and myelin forming glial cell.

Bruton's Tyrosine Kinase Inhibition Promotes Myelin Repair.

BACKGROUND: Microglia are the resident macrophages of the central nervous system (CNS). In multiple sclerosis (MS) and related experimental models, microglia have either a pro-inflammatory or a pro-regenerative/pro-remyelinating function. Inhibition of Bruton's tyrosine kinase (BTK), a member of the Tec family of kinases, has been shown to block differentiation of pro-inflammatory macrophages in response to granulocyte-macrophage colony-stimulating factor in vitro. However, the role of BTK in the CNS is unknown. METHODS: Our aim was to investigate the effect of BTK inhibition on myelin repair in ex vivo and in vivo experimental models of demyelination and remyelination. The remyelination effect of a BTK inhibitor (BTKi; BTKi-1) was then investigated in LPC-induced demyelinated cerebellar organotypic slice cultures and metronidazole-induced demyelinated Xenopus MBP-GFP-NTR transgenic tadpoles. RESULTS: Cellular detection of BTK and its activated form BTK-phospho-Y223 (p-BTK) was determined by immunohistochemistry in organotypic cerebellar slice cultures, before and after lysophosphatidylcholine (LPC)-induced demyelination. A low BTK signal detected by immunolabeling under normal conditions in cerebellar slices was in sharp contrast to an 8.5-fold increase in the number of BTK-positive cells observed in LPC-demyelinated slice cultures. Under both conditions, approximately 75% of cells expressing BTK and p-BTK were microglia and 25% were astrocytes. Compared with spontaneous recovery, treatment of demyelinated slice cultures and MTZ-demyelinated transgenic tadpoles with BTKi resulted in at least a 1.7-fold improvement of remyelination. CONCLUSION: Our data demonstrate that BTK inhibition is a promising therapeutic strategy for myelin repair.

Myelin replacement triggered by single-cell demyelination in mouse cortex.

Myelin, rather than being a static insulator of axons, is emerging as an active participant in circuit plasticity. This requires precise regulation of oligodendrocyte numbers and myelination patterns. Here, by devising a laser ablation approach of single oligodendrocytes, followed by in vivo imaging and correlated ultrastructural reconstructions, we report that in mouse cortex demyelination as subtle as the loss of a single oligodendrocyte can trigger robust cell replacement and remyelination timed by myelin breakdown. This results in reliable reestablishment of the original myelin pattern along continuously myelinated axons, while in parallel, patchy isolated internodes emerge on previously unmyelinated axons. Therefore, in mammalian cortex, internodes along partially myelinated cortical axons are typically not reestablished, suggesting that the cues that guide patchy myelination are not preserved through cycles of de- and remyelination. In contrast, myelin sheaths forming continuous patterns show remarkable homeostatic resilience and remyelinate with single axon precision.

Remyelination in multiple sclerosis: from basic science to clinical translation.

The treatment of multiple sclerosis has been transformed by the successful development of immunotherapies that efficiently reduce disease activity and related clinical relapses during the relapsing-remitting phase of the disease. However, the prevention of disability progression, which is due to axonal and neuronal damage and loss, has yet to be achieved and is therapeutically challenging, particularly during the progressive phase of the disease. One strategy to counteract neurodegeneration is to promote neuroprotection by enhancing myelin regeneration, hence restoring nerve conduction and metabolic support to the axon. Animal studies have provided targets for interventions to improve brain and spinal cord remyelination, paving the way for the translation of this research to humans. From these initial and promising forays, further problems have emerged, including questions on how best to design these clinical trials and appropriately measure the outcomes. Solving these problems will need additional work before efficacious pro-remyelination therapies will be ready for people with multiple sclerosis, but there is a real sense of hope that researchers are getting closer to a successful therapy.

The ENDpoiNTs Project: Novel Testing Strategies for Endocrine Disruptors Linked to Developmental Neurotoxicity.

Ubiquitous exposure to endocrine-disrupting chemicals (EDCs) has caused serious concerns about the ability of these chemicals to affect neurodevelopment, among others. Since endocrine disruption (ED)-induced developmental neurotoxicity (DNT) is hardly covered by the chemical testing tools that are currently in regulatory use, the Horizon 2020 research and innovation action ENDpoiNTs has been launched to fill the scientific and methodological gaps related to the assessment of this type of chemical toxicity. The ENDpoiNTs project will generate new knowledge about ED-induced DNT and aims to develop and improve in vitro, in vivo, and in silico models pertaining to ED-linked DNT outcomes for chemical testing. This will be achieved by establishing correlative and causal links between known and novel neurodevelopmental endpoints and endocrine pathways through integration of molecular, cellular, and organismal data from in vitro and in vivo models. Based on this knowledge, the project aims to provide adverse outcome pathways (AOPs) for ED-induced DNT and to develop and integrate new testing tools with high relevance for human health into European and international regulatory frameworks.

Problems and Pitfalls of Identifying Remyelination in Multiple Sclerosis.

Regenerative medicines that promote remyelination in multiple sclerosis (MS) are making the transition from laboratory to clinical trials. While animal models provide the experimental flexibility to analyze mechanisms of remyelination, here we discuss the challenges in understanding where and how remyelination occurs in MS.

Role of a Contactin multi-molecular complex secreted by oligodendrocytes in nodal protein clustering in the CNS.

The fast and reliable propagation of action potentials along myelinated fibers relies on the clustering of voltage-gated sodium channels at nodes of Ranvier. Axo-glial communication is required for assembly of nodal proteins in the central nervous system, yet the underlying mechanisms remain poorly understood. Oligodendrocytes are known to support node of Ranvier assembly through paranodal junction formation. In addition, the formation of early nodal protein clusters (or prenodes) along axons prior to myelination has been reported, and can be induced by oligodendrocyte conditioned medium (OCM). Our recent work on cultured hippocampal neurons showed that OCM-induced prenodes are associated with an increased conduction velocity (Freeman et al., 2015). We here unravel the nature of the oligodendroglial secreted factors. Mass spectrometry analysis of OCM identified several candidate proteins (i.e., Contactin-1, ChL1, NrCAM, Noelin2, RPTP/Phosphacan, and Tenascin-R). We show that Contactin-1 combined with RPTP/Phosphacan or Tenascin-R induces clusters of nodal proteins along hippocampal GABAergic axons. Furthermore, Contactin-1-immunodepleted OCM or OCM from Cntn1-null mice display significantly reduced clustering activity, that is restored by addition of soluble Contactin-1. Altogether, our results identify Contactin-1 secreted by oligodendrocytes as a novel factor that may influence early steps of nodal sodium channel cluster formation along specific axon populations.

Conditional Demyelination and Remyelination in a Transgenic Xenopus laevis.

Multiple sclerosis (MS) is the first cause of acquired disability progression in the young adult. Pathology of MS associates inflammation, demyelination, and neurodegeneration. The development of immunotherapies, by reducing the relapse rate, has profoundly impacted short-term prognosis and patients' quality of life. These anti-inflammatory medications, however, have not proven to be sufficient to prevent long-term disability progression, resulting from axonal transection and neuronal damage, consequences of prolonged demyelination. Promoting remyelination is therefore a key therapeutic strategy to limit handicap progression, and represent the major therapeutic challenge in MS. Here we present a simple, rapid, and cost-effective experimental model developed in Xenopus laevis to screen in vivo molecules promoting remyelination.

Oligodendrocyte precursor survival and differentiation requires chromatin remodeling by Chd7 and Chd8.

Oligodendrocyte precursor cells (OPCs) constitute the main proliferative cells in the adult brain, and deregulation of OPC proliferation-differentiation balance results in either glioma formation or defective adaptive (re)myelination. OPC differentiation requires significant genetic reprogramming, implicating chromatin remodeling. Mounting evidence indicates that chromatin remodelers play important roles during normal development and their mutations are associated with neurodevelopmental defects, with CHD7 haploinsuficiency being the cause of CHARGE syndrome and CHD8 being one of the strongest autism spectrum disorder (ASD) high-risk-associated genes. Herein, we report on uncharacterized functions of the chromatin remodelers Chd7 and Chd8 in OPCs. Their OPC-chromatin binding profile, combined with transcriptome and chromatin accessibility analyses of Chd7-deleted OPCs, demonstrates that Chd7 protects nonproliferative OPCs from apoptosis by chromatin closing and transcriptional repression of p53 Furthermore, Chd7 controls OPC differentiation through chromatin opening and transcriptional activation of key regulators, including Sox10, Nkx2.2, and Gpr17 However, Chd7 is dispensable for oligodendrocyte stage progression, consistent with Chd8 compensatory function, as suggested by their common chromatin-binding profiles and genetic interaction. Finally, CHD7 and CHD8 bind in OPCs to a majority of ASD risk-associated genes, suggesting an implication of oligodendrocyte lineage cells in ASD neurological defects. Our results thus offer new avenues to understand and modulate the CHD7 and CHD8 functions in normal development and disease.

The Xenopus tadpole: An in vivo model to screen drugs favoring remyelination.

BACKGROUND: In multiple sclerosis, development of screening tools for remyelination-promoting molecules is timely. OBJECTIVE: A Xenopus transgenic line allowing conditional ablation of myelinating oligodendrocytes has been adapted for in vivo screening of remyelination-favoring molecules. METHODS: In this transgenic, the green fluorescent protein reporter is fused to E. coli nitroreductase and expressed specifically in myelinating oligodendrocytes. Nitroreductase converts the innocuous pro-drug metronidazole to a cytotoxin. Spontaneous remyelination occurs after metronidazole-induced demyelinating responses. As tadpoles are transparent, these events can be monitored in vivo and quantified. At the end of metronidazole-induced demyelination, tadpoles were screened in water containing the compounds tested. After 72 h, remyelination was assayed by counting numbers of oligodendrocytes per optic nerve. RESULTS: Among a battery of molecules tested, siponimod, a dual agonist of sphingosine-1-phosphate receptor 1 and 5, was among the most efficient favoring remyelination. Crispr/cas9 gene editing showed that the promyelinating effect of siponimod involves the sphingosine-1-phosphate receptor 5. CONCLUSION: This Xenopus transgenic line constitutes a simple in vivo screening platform for myelin repair therapeutics. We validated several known promyelinating compounds and demonstrated that the strong remyelinating efficacy of siponimod implicates the sphingosine-1-phosphate receptor 5.

Transient hypothyroidism favors oligodendrocyte generation providing functional remyelination in the adult mouse brain.

In the adult brain, both neurons and oligodendrocytes can be generated from neural stem cells located within the Sub-Ventricular Zone (SVZ). Physiological signals regulating neuronal versus glial fate are largely unknown. Here we report that a thyroid hormone (T3)-free window, with or without a demyelinating insult, provides a favorable environment for SVZ-derived oligodendrocyte progenitor generation. After demyelination, oligodendrocytes derived from these newly-formed progenitors provide functional remyelination, restoring normal conduction. The cellular basis for neuronal versus glial determination in progenitors involves asymmetric partitioning of EGFR and TRα1, expression of which favor glio- and neuro-genesis, respectively. Moreover, EGFR+ oligodendrocyte progenitors, but not neuroblasts, express high levels of a T3-inactivating deiodinase, Dio3. Thus, TRα absence with high levels of Dio3 provides double-pronged blockage of T3 action during glial lineage commitment. These findings not only transform our understanding of how T3 orchestrates adult brain lineage decisions, but also provide potential insight into demyelinating disorders.