Delphine Meffre

Lithium enhances remyelination of peripheral nerves

Glycogen synthase kinase 3β (GSK3β) inhibitors, especially the mood stabilizer lithium chloride, are also used as neuroprotective or anti-inflammatory agents. We studied the influence of LiCl on the remyelination of peripheral nerves. We showed that the treatment of adult mice with LiCl after facial nerve crush injury stimulated the expression of myelin genes, restored the myelin structure, and accelerated the recovery of whisker movements. LiCl treatment also promoted remyelination of the sciatic nerve after crush. We also demonstrated that peripheral myelin gene MPZ and PMP22 promoter activities, transcripts, and protein levels are stimulated by GSK3β inhibitors (LiCl and SB216763) in Schwann cells as well as in sciatic and facial nerves. LiCl exerts its action in Schwann cells by increasing the amount of β-catenin and provoking its nuclear localization. We showed by ChIP experiments that LiCl treatment drives β-catenin to bind to T-cell factor/lymphoid-enhancer factor response elements identified in myelin genes. Taken together, our findings open perspectives in the treatment of nerve demyelination by administering GSK3β inhibitors such as lithium.

Interplay between LXR and Wnt/β-Catenin Signaling in the Negative Regulation of Peripheral Myelin Genes by Oxysterols

Oxysterols are reactive molecules generated from the oxidation of cholesterol. Their implication in cholesterol homeostasis and in the progression of neurodegenerative disorders is well known, but few data are available for their functions in the peripheral nervous system. Our aim was to study the influence of oxysterols on myelin gene expression and myelin sheath formation in peripheral nerves. We show by gas chromatography/mass spectrometry that Schwann cells and sciatic nerves contain 24(S)-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol and that they express their biosynthetic enzymes and receptors (liver X receptors LXRα and LXRβ). We demonstrate that oxysterols inhibit peripheral myelin gene expression [myelin protein zero (MPZ) and peripheral myelin protein-22 (PMP22)] in a Schwann cell line. This downregulation is mediated by either LXRα or LXRβ, depending on the promoter context, as suggested by siRNA strategy and chromatin immunoprecipitation assays in Schwann cells and in the sciatic nerve of LXR knock-out mice. Importantly, the knock-out of LXR in mice results in thinner myelin sheaths surrounding the axons. Oxysterols repress myelin genes via two mechanisms: by binding of LXRs to myelin gene promoters and by inhibiting the Wnt/β-catenin pathway that is crucial for the expression of myelin genes. The Wnt signaling components (Disheveled, TCF/LEF, β-catenin) are strongly repressed by oxysterols. Furthermore, the recruitment of β-catenin at the levels of the MPZ and PMP22 promoters is decreased. Our data reveal new endogenous mechanisms for the negative regulation of myelin gene expression, highlight the importance of oxysterols and LXR in peripheral nerve myelination, and open new perspectives of treating demyelinating diseases with LXR agonists.

Liver X receptors alpha and beta promote myelination and remyelination in the cerebellum.

Significance Liver X receptors (LXRs) α and β are the two major receptors of oxysterols, oxygenated derivatives of cholesterol. They control the homeostasis of cholesterol, an important lipid constituent of myelin sheaths. In the central nervous system, these insulating structures are generated by oligodendrocytes and are stabilized by myelin proteins. Here, we provide evidence of a new role of LXRs in the myelin physiology of the cerebellum. Mice invalidated for both LXRs exhibit alteration in motor coordination and spatial learning linked with myelination deficits. We demonstrated that LXRs intervene both in oligodendroglial cell maturation and in the transcriptional control of myelin gene expression during (re)myelination processes. The identification of new pathways governing myelination provides innovative avenues for remyelination. Liver X receptors (LXRs) α and β are nuclear receptors activated by oxysterols that originated from the oxidation of cholesterol. They are crucial for cholesterol homeostasis, a major lipid constituent of myelin sheaths that are formed by oligodendrocytes. However, the role of LXRs in myelin generation and maintenance is poorly understood. Here, we show that LXRs are involved in myelination and remyelination processes. LXRs and their ligands are present in oligodendrocytes. We found that mice invalidated for LXRs exhibit altered motor coordination and spatial learning, thinner myelin sheaths, and reduced myelin gene expression. Conversely, activation of LXRs by either 25-hydroxycholesterol or synthetic TO901317 stimulates myelin gene expression at the promoter, mRNA, and protein levels, directly implicating LXRα/β in the transcriptional control of myelin gene expression. Interestingly, activation of LXRs also promotes oligodendroglial cell maturation and remyelination after lysolecithin-induced demyelination of organotypic cerebellar slice cultures. Together, our findings represent a conceptual advance in the transcriptional control of myelin gene expression and strongly support a new role of LXRs as positive modulators in central (re)myelination processes.

Lithium chloride stimulates PLP and MBP expression in oligodendrocytes via Wnt/β-catenin and Akt/CREB pathways

Deciphering the molecular pathways involved in myelin gene expression is a major point of interest to better understand re/myelination processes. In this study, we investigated the role of Lithium Chloride (LiCl), a drug largely used for the treatment of neurological disorders, on the two major central myelin gene expression (PLP and MBP) in mouse oligodendrocytes. We show that LiCl enhances the expression of both PLP and MBP, by increasing mRNA amount and promoter activities. We investigated whether Wnt/β-catenin and/or Akt/CREB pathways are modulated by LiCl to regulate myelin gene expression. We showed that β-catenin is required both for PLP and MBP basal promoter activities and for LiCl-induced myelin gene stimulation. Furthermore, while CREB functionality does not influence PLP expression, MBP promoter activity depends on Akt/CREB activation. Finally, we show that LiCl can stimulate oligodendrocyte morphological maturation, and promote remyelination after lysolecithin-induced demyelination of organotypic cerebellar slice cultures. Our data provide mechanistic evidences that Akt/CREB together with β-catenin participate in the transcriptional control of PLP and MBP exerted by LiCl. Therefore, the use of LiCl to balance between β-catenin and CREB effectors could be considered as an efficient remyelinating strategy.

Wnt and lithium: a common destiny in the therapy of nervous system pathologies?

Wnt signaling is required for neurogenesis, the fate of neural progenitors, the formation of neuronal circuits during development, neuron positioning and polarization, axon and dendrite development and finally for synaptogenesis. This signaling pathway is also implicated in the generation and differentiation of glial cells. In this review, we describe the mechanisms of action of Wnt signaling pathways and their implication in the development and correct functioning of the nervous system. We also illustrate how a dysregulated Wnt pathway could lead to psychiatric, neurodegenerative and demyelinating pathologies. Lithium, used for the treatment of bipolar disease, inhibits GSK3β, a central enzyme of the Wnt/β-catenin pathway. Thus, lithium could, to some extent, mimic Wnt pathway. We highlight the possible dialogue between lithium therapy and modulation of Wnt pathway in the treatment of the diseases of the nervous system.

Differential regulation of Wnt/beta-catenin signaling by Liver X Receptors in Schwann cells and oligodendrocytes.

Oxysterols are reactive molecules generated by the oxidation of cholesterol. Their implication in cholesterol homeostasis and in the progression of neurodegenerative disorders is well known. Here, we study the role of oxysterols and their nuclear receptors, Liver X Receptor (LXR), in myelinating glial cells of the central and peripheral nervous systems. First, we show by gas chromatography/mass spectrometry that the brain, sciatic nerve, oligodendrocytes and Schwann cells contain 24(S)-hydroxycholesterol, 25-hydroxycholesterol (25-OH) and 27-hydroxycholesterol, and they express their biosynthetic enzymes. We observed a differential effect of 25-OH toward myelin genes (MPZ and PMP22) expression: 25-OH inhibits MPZ and PMP22 in Schwann cell line but not in oligodendrocyte cell line. Importantly, the invalidation of LXR in mice enhanced MPZ and PMP22 transcripts expression in the sciatic nerve, but inhibited their expression in the brain. We have previously reported that Wnt signaling pathway is crucial for myelin gene expression. We show that the transcripts of Wnt components (Disheveled, TCF3, beta-catenin) are strongly repressed by oxysterols in Schwann cells but are activated in oligodendrocytes. Furthermore, we show by immunofluorescent labeling that beta-catenin is re-localized on the level of the Golgi apparatus of Schwann cells after incubation with 25-OH. We did not observe such an unusual localization of beta-catenin in oligodendrocytes. Our findings reveal a complex cross-talk between LXR and Wnt/beta-catenin pathway in myelinating glial cells.

Involvement of Aryl hydrocarbon receptor in myelination and in human nerve sheath tumorigenesis

Significance Aryl hydrocarbon receptor (AHR) is well known to mediate xenobiotic metabolism in vertebrates. Growing evidence reveals that AHR seems to have endogenous roles in the development and functioning of different organs. In our current study, we describe a role of AHR in peripheral myelination and in nerve sheath tumors. We show that the AHR pathway is dysregulated in human biopsies of nerve tumors. The blockade of AHR provokes cell death in nerve tumors, suggesting a therapeutic avenue in the treatment of this invasive cancer. Furthermore, the inhibition of Ahr in mice provokes locomotor defects and alteration of myelin structure. This work unravels an endogenous role of Ahr in peripheral myelination and a potential treatment of nerve tumours. Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor involved in xenobiotic metabolism. Plexiform neurofibromas (PNFs) can transform into malignant peripheral nerve sheath tumors (MPNSTs) that are resistant to existing therapies. These tumors are primarily composed of Schwann cells. In addition to neurofibromatosis type 1 (NF1) gene inactivation, further genetic lesions are required for malignant transformation. We have quantified the mRNA expression levels of AHR and its associated genes in 38 human samples. We report that AHR and the biosynthetic enzymes of its endogenous ligand are overexpressed in human biopsies of PNFs and MPNSTs. We also detect a strong nuclear AHR staining in MPNSTs. The inhibition of AHR by siRNA or antagonists, CH-223191 and trimethoxyflavone, induces apoptosis in human MPNST cells. Since AHR dysregulation is observed in these tumors, we investigate AHR involvement in Schwann cell physiology. Hence, we studied the role of AHR in myelin structure and myelin gene regulation in Ahr−/− mice during myelin development. AHR ablation leads to locomotion defects and provokes thinner myelin sheaths around the axons. We observe a dysregulation of myelin gene expression and myelin developmental markers in Ahr−/− mice. Interestingly, AHR does not directly bind to myelin gene promoters. The inhibition of AHR in vitro and in vivo increased β-catenin levels and stimulated the binding of β-catenin on myelin gene promoters. Taken together, our findings reveal an endogenous role of AHR in peripheral myelination and in peripheral nerve sheath tumors. Finally, we suggest a potential therapeutic approach by targeting AHR in nerve tumors.

Liver X Receptors differentially modulate central myelin gene mRNA levels in a region-, age- and isoform-specific manner

Liver X Receptors (LXRs) α and β are nuclear receptors able to bind oxidative forms of cholesterol. They play important roles in the central nervous system (CNS), through their implication in a large variety of physiological and pathological processes among which modulation of cholesterol homeostasis and inflammation. Besides, we recently revealed their crucial role in myelination and remyelination in the cerebellum. Given the pleiotropic effects of such receptors on CNS functioning, we studied here the influence of LXRs on myelin gene mRNA accumulation in the major myelinated regions of the CNS in vivo. We show that both LXR isoforms differentially affect mRNA amount of myelin genes (PLP and MBP) in highly myelinated structures such as spinal cord, corpus callosum, optic nerve and cerebellum. In the adult, LXR activation by the synthetic agonist TO901317 significantly increases myelin gene mRNA amount in the cerebellum but not in the other regions studied. Invalidation of the sole LXRβ isoform leads to decreased PLP and MBP mRNA levels in all the structures except the spinal cord, while the knock out of both isoforms (LXR dKO) decreases myelin gene mRNA amounts in all the regions tested except the corpus callosum. Interestingly, during myelination process (post-natal day 21), both cerebellum and optic nerve display a decrease in myelin gene mRNA levels in LXR dKO mice. Concomitantly, PLP and MBP mRNA accumulation in the spinal cord is increased. Relative expression level of LXR isoforms could account for the differential modulation of myelin gene expression in the CNS. Altogether our results suggest that, within the CNS, each LXR isoform differentially influences myelin gene mRNA levels in a region- and age-dependant manner, participating in the fine regulation of myelin gene expression.

Targeting demyelination via α-secretases promoting sAPPα release to enhance remyelination in central nervous system.

Remyelination is an endogenous regenerative process of myelin repair in the central nervous system (CNS) with limited efficacy in demyelinating disorders. As strategies enhancing endogenous remyelination become a therapeutic challenge, we have focused our study on α-secretase-induced sAPPα release, a soluble endogenous protein with neuroprotective and neurotrophic properties. However, the role of sAPPα in remyelination is not known. Therefore, we investigated the remyelination potential of α-secretase-induced sAPPα release following CNS demyelination in mice. Acute demyelination was induced by feeding mice with cuprizone (CPZ) for 5weeks. To test the protective effect and the remyelination potential of etazolate, an α-secretase activator, we designed two treatment protocols. Etazolate was administrated either during the last two weeks or at the end of the CPZ intoxication. In both protocols, etazolate restored the number of myelinated axons in corpus callosum with a corresponding increase in the amount of MBP, one of the major myelin proteins in the brain. We also performed ex vivo studies to decipher etazolates mechanism of action in a lysolecithin-induced demyelination model using organotypic culture of cerebellar slices. Etazolate treatment was able to i) enhance the release of sAPPα in the culture media of demyelinated slices, ii) protect myelinated axons from demyelination, iii) increase the number of mature oligodendrocytes, iv) promote the reappearance of the paired Caspr+ adjacent to the nodes of Ranvier and v) increase the percentage of myelinated axons with short internodes, an indicator of remyelination. Etazolate failed to promote all the aforementioned effects in the presence of GI254023X, an α-secretase inhibitor. Moreover, the protective effects of etazolate in demyelinated slices were mimicked by sAPPα treatment in a dose-dependent manner. In conclusion, etazolate-induced sAPPα release protects myelinated axons from demyelination while also promoting remyelination. This work, thus, highlights the therapeutic potential of strategies that enhance sAPPα release in demyelinating disorders.

Mechanical Stretch of High Magnitude Provokes Axonal Injury, Elongation of Paranodal Junctions, and Signaling Alterations in Oligodendrocytes

Increasing findings suggest that demyelination may play an important role in the pathophysiology of brain injury, but the exact mechanisms underlying such damage are not well known. Mechanical tensile strain of brain tissue occurs during traumatic brain injury. Several studies have investigated the cellular and molecular events following a static tensile strain of physiological magnitude on individual cells such as oligodendrocytes. However, the pathobiological impact of high-magnitude mechanical strain on oligodendrocytes and myelinated fibers remains under investigated. In this study, we reported that an applied mechanical tensile strain of 30% on mouse organotypic culture of cerebellar slices induced axonal injury and elongation of paranodal junctions, two hallmarks of brain trauma. It was also able to activate MAPK-ERK1/2 signaling, a stretch-induced responsive pathway. The same tensile strain applied to mouse oligodendrocytes in primary culture induced a profound damage to cell morphology, partial cell loss, and a decrease of myelin protein expression. The lower tensile strain of 20% also caused cell loss and the remaining oligodendrocytes appeared retracted with decreased myelin protein expression. Finally, high-magnitude tensile strain applied to 158N oligodendroglial cells altered myelin protein expression, dampened MAPK-ERK1/2 and MAPK-p38 signaling, and enhanced the production of reactive oxygen species. The latter was accompanied by increased protein oxidation and an alteration of anti-oxidant defense that was strain magnitude-dependent. In conclusion, mechanical stretch of high magnitude provokes axonal injury with significant alterations in oligodendrocyte biology that could initiate demyelination.