Physiological aging and inflammation-induced cellular senescence may contribute to oligodendroglial dysfunction in MS.

Aging affects all cell types in the CNS and plays an important role in CNS diseases. However, the underlying molecular mechanisms driving these age-associated changes and their contribution to diseases are only poorly understood. The white matter in the aging brain as well as in diseases, such as Multiple sclerosis is characterized by subtle abnormalities in myelin sheaths and paranodes, suggesting that oligodendrocytes, the myelin-maintaining cells of the CNS, lose the capacity to preserve a proper myelin structure and potentially function in age and certain diseases. Here, we made use of directly converted oligodendrocytes (dchiOL) from young, adult and old human donors to study age-associated changes. dchiOL from all three age groups differentiated in an comparable manner into O4 + immature oligodendrocytes, but the proportion of MBP + mature dchiOL decreased with increasing donor age. This was associated with an increased ROS production and upregulation of cellular senescence markers such as CDKN1A, CDKN2A in old dchiOL. Comparison of the transcriptomic profiles of dchiOL from adult and old donors revealed 1324 differentially regulated genes with limited overlap with transcriptomic profiles of the donors' fibroblasts or published data sets from directly converted human neurons or primary rodent oligodendroglial lineage cells. Methylome analyses of dchiOL and human white matter tissue samples demonstrate that chronological and epigenetic age correlate in CNS white matter as well as in dchiOL and resulted in the identification of an age-specific epigenetic signature. Furthermore, we observed an accelerated epigenetic aging of the myelinated, normal appearing white matter of multiple sclerosis (MS) patients compared to healthy individuals. Impaired differentiation and upregulation of cellular senescence markers could be induced in young dchiOL in vitro using supernatants from pro-inflammatory microglia. In summary, our data suggest that physiological aging as well as inflammation-induced cellular senescence contribute to oligodendroglial pathology in inflammatory demyelinating diseases such as MS.

Influx of T cells into corpus callosum increases axonal injury, but does not change the course of remyelination in toxic demyelination.

Multiple sclerosis (MS) is a focal inflammatory and demyelinating disease. The inflammatory infiltrates consist of macrophages/microglia, T and B cells. Remyelination (RM) is an endogenous repair process which frequently fails in MS patients. In earlier studies, T cells either promoted or impaired RM. Here, we used the combined cuprizone/MOG-EAE model to further dissect the functional role of T cells for RM. The combination of MOG immunization with cuprizone feeding targeted T cells to the corpus callosum and increased the extent of axonal injury. Global gene expression analyses demonstrated significant changes in the inflammatory environment; however, additional MOG immunization did not alter the course of RM. Our results suggest that the inflammatory environment in the combined model affects axons and oligodendrocytes differently and that oligodendroglial lineage cells might be less susceptible to T cell mediated injury.

Modulation of pacemaker channel function in a model of thalamocortical hyperexcitability by demyelination and cytokines.

A consensus is yet to be reached regarding the exact prevalence of epileptic seizures or epilepsy in multiple sclerosis (MS). In addition, the underlying pathophysiological basis of the reciprocal interaction among neuroinflammation, demyelination, and epilepsy remains unclear. Therefore, a better understanding of cellular and network mechanisms linking these pathologies is needed. Cuprizone-induced general demyelination in rodents is a valuable model for studying MS pathologies. Here, we studied the relationship among epileptic activity, loss of myelin, and pro-inflammatory cytokines by inducing acute, generalized demyelination in a genetic mouse model of human absence epilepsy, C3H/HeJ mice. Both cellular and network mechanisms were studied using in vivo and in vitro electrophysiological techniques. We found that acute, generalized demyelination in C3H/HeJ mice resulted in a lower number of spike-wave discharges, increased cortical theta oscillations, and reduction of slow rhythmic intrathalamic burst activity. In addition, generalized demyelination resulted in a significant reduction in the amplitude of the hyperpolarization-activated inward current (Ih) in thalamic relay cells, which was accompanied by lower surface expression of hyperpolarization-activated, cyclic nucleotide-gated channels, and the phosphorylated form of TRIP8b (pS237-TRIP8b). We suggest that demyelination-related changes in thalamic Ih may be one of the factors defining the prevalence of seizures in MS.

Dimethyl fumarate treatment restrains the antioxidative capacity of T cells to control autoimmunity.

Dimethyl fumarate, an approved treatment for relapsing-remitting multiple sclerosis, exerts pleiotropic effects on immune cells as well as CNS resident cells. Here, we show that dimethyl fumarate exerts a profound alteration of the metabolic profile of human CD4+ as well as CD8+ T cells and restricts their antioxidative capacities by decreasing intracellular levels of the reactive oxygen species scavenger glutathione. This causes an increase in mitochondrial reactive oxygen species levels accompanied by an enhanced mitochondrial stress response, ultimately leading to impaired mitochondrial function. Enhanced mitochondrial reactive oxygen species levels not only result in enhanced T-cell apoptosis in vitro as well as in dimethyl fumarate-treated patients, but are key for the well-known immunomodulatory effects of dimethyl fumarate both in vitro and in an animal model of multiple sclerosis, i.e. experimental autoimmune encephalomyelitis. Indeed, dimethyl fumarate immune-modulatory effects on T cells were completely abrogated by pharmacological interference of mitochondrial reactive oxygen species production. These data shed new light on dimethyl fumarate as bona fide immune-metabolic drug that targets the intracellular stress response in activated T cells, thereby restricting mitochondrial function and energetic capacity, providing novel insight into the role of oxidative stress in modulating cellular immune responses and T cell-mediated autoimmunity.

SKAP2 as a new regulator of oligodendroglial migration and myelin sheath formation.

Oligodendroglial progenitor cells (OPCs) are highly proliferative and migratory cells, which differentiate into complex myelin forming and axon ensheathing mature oligodendrocytes during myelination. Recent studies indicate that the oligodendroglial cell population is heterogeneous on transcriptional and functional level depending on the location in the central nervous system. Here, we compared intrinsic properties of OPC from spinal cord and brain on functional and transcriptional level. Spinal cord OPC demonstrated increased migration as well as differentiation capacity. Moreover, transcriptome analysis revealed differential expression of several genes between both OPC populations. In spinal cord OPC, we confirmed upregulation of SKAP2, a cytoplasmatic adaptor protein known for its implication in cytoskeletal remodeling and migration in other cell types. Recent findings suggest that actin dynamics determine not only oligodendroglial migration, but also differentiation: Whereas actin polymerization is important for process extension, actin destabilization and depolymerization is required for myelin sheath formation. Downregulation or complete lack of SKAP2 in OPC resulted in reduced migration and impaired morphological maturation in oligodendrocytes. In contrast, overexpression of SKAP2 as well as constitutively active SKAP2 increased OPC migration suggesting that SKAP2 function is dependent on activation by phosphorylation. Furthermore, lack of SKAP2 enhanced the positive effect on OPC migration after integrin activation suggesting that SKAP2 acts as modulator of integrin dependent migration. In summary, we demonstrate the presence of intrinsic differences between spinal cord and brain OPC and identified SKAP2 as a new regulator of oligodendroglial migration and sheath formation.

One-step Reprogramming of Human Fibroblasts into Oligodendrocyte-like Cells by SOX10, OLIG2, and NKX6.2.

Limited access to human oligodendrocytes impairs better understanding of oligodendrocyte pathology in myelin diseases. Here, we describe a method to robustly convert human fibroblasts directly into oligodendrocyte-like cells (dc-hiOLs), which allows evaluation of remyelination-promoting compounds and disease modeling. Ectopic expression of SOX10, OLIG2, and NKX6.2 in human fibroblasts results in rapid generation of O4+ cells, which further differentiate into MBP+ mature oligodendrocyte-like cells within 16 days. dc-hiOLs undergo chromatin remodeling to express oligodendrocyte markers, ensheath axons, and nanofibers in vitro, respond to promyelination compound treatment, and recapitulate in vitro oligodendroglial pathologies associated with Pelizaeus-Merzbacher leukodystrophy related to PLP1 mutations. Furthermore, DNA methylome analysis provides evidence that the CpG methylation pattern significantly differs between dc-hiOLs derived from fibroblasts of young and old donors, indicating the maintenance of the source cells' "age." In summary, dc-hiOLs represent a reproducible technology that could contribute to personalized medicine in the field of myelin diseases.

Oligodendroglial glycolipids in (Re)myelination: implications for multiple sclerosis research.

Covering: up to 2020 This short review surveys aspects of glycolipid-based natural products and their biological relevance in multiple sclerosis (MS). The role of isolated gangliosides in disease models is discussed together with an overview of ganglioside-inspired small molecule drugs and imaging probes. The discussion is extended to neurodegeneration in a more general context and addresses the need for more efficient synthetic methods to generate (glyco)structures that are of therapeutic relevance.

Total Chemical Syntheses of the GM3 and F-GM3 Ganglioside Epitopes and Comparative Pre-Clinical Evaluation for Non-Invasive Imaging of Oligodendrocyte Differentiation.

Gangliosides are intimately involved in a plenum of (neuro)inflammatory processes, yet progress in establishing structure-function interplay is frequently hindered by the availability of well-defined glycostructures. Motivated by the ubiquity of the ganglioside GM3 in chemical neurology, and in particular by its conspicuous presence in myelin, the GM3 epitope was examined with a view to preclinical validation as a tracer. The suitability of this scaffold for the noninvasive imaging of oligodendrocyte differentiation in Multiple sclerosis is disclosed. The stereocontrolled synthesis of a site-selectively fluorinated analogue (F-GM3) is also disclosed to enable a comparative analysis in oligodendrocyte (OL) differentiation. Whereas the native epitope caused a decrease in the viability in a dose-dependent manner, the addition of distinct F-GM3 concentrations over 48 h had no impact on the OL viability. This is likely a consequence of the enhanced hydrolytic stability imparted by the fluorination and highlights the potential of fluorinated glycostructures in the field of molecular imaging. Given the predominant expression of GM3 in oligodendrocytes and the capacity of GM3 to interact with myelin-associated proteins, this preclinical evaluation has revealed F-GM3 to be an intriguing candidate for neurological imaging.

The K2P -channel TASK1 affects Oligodendroglial differentiation but not myelin restoration.

In multiple sclerosis, demyelination occurs as a consequence of chronic autoimmunity in the central nervous system causing progressive neurological impairment in patients. After a demyelinating event, new myelin sheaths are formed by adult oligodendroglial progenitor cells; a process called remyelination. However, remyelination often fails in multiple sclerosis due to insufficient recruitment and differentiation of oligodendroglial precursor cells. A pivotal role for the two-pore-domain potassium (K2P ) channel, TASK1, has already been proven for an animal model of multiple sclerosis. However, the mechanisms underlying the TASK1-mediated effects are still elusive. Here, we tested the role of TASK1 channels in oligodendroglial differentiation and remyelination after cuprizone-induced demyelination in male mice. We found TASK1 channels to be functionally expressed on primary murine and human, pluripotent stem cell-derived oligodendrocytes. Lack of TASK1 channels resulted in an increase of mature oligodendrocytes in vitro as well as a higher number of mature oligodendrocytes and accelerated developmental myelination in vivo. Mechanistically, Task1-deficient cells revealed a higher amount of phosphorylated WNK1, a kinase known to be involved in the downstream signaling of the myelination regulator LINGO-1. Furthermore, we analyzed the effect of genetic TASK1 ablation or pharmacological TASK1 inhibition on disease-related remyelination. Neither channel inhibition nor lack of TASK1 channels promoted remyelination after pathological demyelination. In summary, we conclude that functional TASK1 channels participate in the modulation of differentiating oligodendroglial cells in a previously unknown manner. However, while being involved in developmental myelination our data suggest that TASK1 channels have no major effect on remyelination.

Protective potential of dimethyl fumarate in a mouse model of thalamocortical demyelination.

Alterations in cortical cellular organization, network functionality, as well as cognitive and locomotor deficits were recently suggested to be pathological hallmarks in multiple sclerosis and corresponding animal models as they might occur following demyelination. To investigate functional changes following demyelination in a well-defined, topographically organized neuronal network, in vitro and in vivo, we focused on the primary auditory cortex (A1) of mice in the cuprizone model of general de- and remyelination. Following myelin loss in this model system, the spatiotemporal propagation of incoming stimuli in A1 was altered and the hierarchical activation of supra- and infragranular cortical layers was lost suggesting a profound effect exerted on neuronal network level. In addition, the response latency in field potential recordings and voltage-sensitive dye imaging was increased following demyelination. These alterations were accompanied by a loss of auditory discrimination abilities in freely behaving animals, a reduction of the nuclear factor-erythroid 2-related factor-2 (Nrf-2) protein in the nucleus in histological staining and persisted during remyelination. To find new strategies to restore demyelination-induced network alteration in addition to the ongoing remyelination, we tested the cytoprotective potential of dimethyl fumarate (DMF). Therapeutic treatment with DMF during remyelination significantly modified spatiotemporal stimulus propagation in the cortex, reduced the cognitive impairment, and prevented the demyelination-induced decrease in nuclear Nrf-2. These results indicate the involvement of anti-oxidative mechanisms in regulating spatiotemporal cortical response pattern following changes in myelination and point to DMF as therapeutic compound for intervention.

Publisher Correction: Nfat/calcineurin signaling promotes oligodendrocyte differentiation and myelination by transcription factor network tuning.

The originally published version of this Article omitted Tanja Kuhlmann and Michael Wegner as jointly supervising authors. This has now been corrected in both the PDF and HTML versions of the Article.

Nfat/calcineurin signaling promotes oligodendrocyte differentiation and myelination by transcription factor network tuning.

Oligodendrocytes produce myelin for rapid transmission and saltatory conduction of action potentials in the vertebrate central nervous system. Activation of the myelination program requires several transcription factors including Sox10, Olig2, and Nkx2.2. Functional interactions among them are poorly understood and important components of the regulatory network are still unknown. Here, we identify Nfat proteins as Sox10 targets and regulators of oligodendroglial differentiation in rodents and humans. Overall levels and nuclear fraction increase during differentiation. Inhibition of Nfat activity impedes oligodendrocyte differentiation in vitro and in vivo. On a molecular level, Nfat proteins cooperate with Sox10 to relieve reciprocal repression of Olig2 and Nkx2.2 as precondition for oligodendroglial differentiation and myelination. As Nfat activity depends on calcium-dependent activation of calcineurin signaling, regulatory network and oligodendroglial differentiation become sensitive to calcium signals. NFAT proteins are also detected in human oligodendrocytes, downregulated in active multiple sclerosis lesions and thus likely relevant in demyelinating disease.

Single Site Fluorination of the GM4 Ganglioside Epitope Upregulates Oligodendrocyte Differentiation.

Relapsing multiple sclerosis is synonymous with demyelination, and thus, suppressing and or reversing this process is of paramount clinical significance. While insulating myelin sheath has a large lipid composition (ca. 70-80%), it also has a characteristically large composition of the sialosylgalactosylceramide gangliosde GM4 present. In this study, the effect of the carbohydrate epitope on oligodendrocyte differentiation is determined. While the native epitope had no impact on oligodendroglial cell viability, a single site OH → F substitution is the structural basis of a significant increase in ATP production that is optimal at 50 µg/mL. From a translational perspective, this subtle change increases the amount of MBP+ oligodendrocytes compared to the control studies and may open up novel therapeutic remyelination strategies.

Activation of FXR pathway does not alter glial cell function.

BACKGROUND: The nuclear receptor farnesoid-X-receptor (FXR; NR1H4) is expressed not only in the liver, gut, kidney and adipose tissue but also in the immune cells. FXR has been shown to confer protection in several animal models of inflammation, including experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). FXR agonists are currently tested in clinical trials for treatment of human metabolic diseases. The beneficial effect of FXR agonists in EAE suggests that FXR might represent a potential target in inflammatory-demyelinating CNS diseases, such as MS. In MS, oligodendrocytes not only undergo cell death but also contribute to remyelination. This repair mechanism is impaired due to a differentiation block of oligodendroglial progenitor cells. Activation of other nuclear receptors that heterodimerize with FXR promote oligodendroglial differentiation. Therefore, we wanted to address the functional relevance of FXR for glial cells, especially for oligodendroglial differentiation. METHODS: We isolated primary murine oligodendrocytes from FXR-deficient (FXR Ko) and wild-type (WT) mice and determined the effect of FXR deficiency and activation on oligodendroglial differentiation by analysing markers of oligodendroglial progenitor cells (OPCs) and mature oligodendrocytes (OLs) using qRT-PCR and immunocytochemistry. Additionally, we determined whether FXR activation modulates the pro-inflammatory profile of astrocytes or microglia and whether this may subsequently modulate oligodendroglial differentiation. These in vitro studies were complemented by histological analyses of oligodendrocytes in FXR Ko mice. RESULTS: FXR is expressed by OPCs and mature oligodendrocytes. However, lack of FXR did not affect oligodendroglial differentiation in vitro or in vivo. Furthermore, activation of FXR using the synthetic agonist GW4064 did not affect oligodendroglial differentiation, remyelination in an ex vivo model or the expression of pro-inflammatory molecules in astrocytes or microglia. Concordantly, no effects of supernatants from macrophages cultured in the presence of GW4064 were observed regarding a possible indirect impact on oligodendroglial differentiation. CONCLUSIONS: Our data suggest that FXR is dispensable for oligodendroglial differentiation and that FXR agonists, such as GW4064, represent a potential therapeutic approach for MS which specifically targets peripheral immune cells including macrophages but not brain-resident cells, such as oligodendrocytes, astrocytes or microglia.

Non-steroidal anti-inflammatory drug indometacin enhances endogenous remyelination.

Multiple sclerosis is the most frequent demyelinating disease in the CNS that is characterized by inflammatory demyelinating lesions and axonal loss, the morphological correlate of permanent clinical disability. Remyelination does occur, but is limited especially in chronic disease stages. Despite effective immunomodulatory therapies that reduce the number of relapses the progressive disease phase cannot be prevented. Therefore, promotion of neuroprotective and repair mechanisms, such as remyelination, represents an attractive additional treatment strategy. A number of pathways have been identified that may contribute to impaired remyelination in MS lesions, among them the Wnt/ß-catenin pathway. Here, we demonstrate that indometacin, a non-steroidal anti-inflammatory drug (NSAID) that has been also shown to modulate the Wnt/ß-catenin pathway in colorectal cancer cells promotes differentiation of primary human and murine oligodendrocytes, myelination of cerebellar slice cultures and remyelination in cuprizone-induced demyelination. Our in vitro experiments using GSK3ß inhibitors, luciferase reporter assays and oligodendrocytes expressing a mutant, dominant stable ß-catenin indicate that the mechanism of action of indometacin depends on GSK3ß activity and ß-catenin phosphorylation. Indometacin might represent a promising treatment option to enhance endogenous remyelination in MS patients.

Nuclear export inhibitors avert progression in preclinical models of inflammatory demyelination.

Axonal damage has been associated with aberrant protein trafficking. We examined a newly characterized class of compounds that target nucleo-cytoplasmic shuttling by binding to the catalytic groove of the nuclear export protein XPO1 (also known as CRM1, chromosome region maintenance protein 1). Oral administration of reversible CRM1 inhibitors in preclinical murine models of demyelination significantly attenuated disease progression, even when started after the onset of paralysis. Clinical efficacy was associated with decreased proliferation of immune cells, characterized by nuclear accumulation of cell cycle inhibitors, and preservation of cytoskeletal integrity even in demyelinated axons. Neuroprotection was not limited to models of demyelination, but was also observed in another mouse model of axonal damage (that is, kainic acid injection) and detected in cultured neurons after knockdown of Xpo1, the gene encoding CRM1. A proteomic screen for target molecules revealed that CRM1 inhibitors in neurons prevented nuclear export of molecules associated with axonal damage while retaining transcription factors modulating neuroprotection.

Chromatin landscape defined by repressive histone methylation during oligodendrocyte differentiation.

In many cell types, differentiation requires an interplay between extrinsic signals and transcriptional changes mediated by repressive and activating histone modifications. Oligodendrocyte progenitors (OPCs) are electrically responsive cells receiving synaptic input. The differentiation of these cells into myelinating oligodendrocytes is characterized by temporal waves of gene repression followed by activation of myelin genes and progressive decline of electrical responsiveness. In this study, we used chromatin isolated from rat OPCs and immature oligodendrocytes, to characterize the genome-wide distribution of the repressive histone marks, H3K9me3 and H3K27me3, during differentiation. Although both marks were present at the OPC stage, only H3K9me3 marks (but not H3K27me3) were found to be increased during differentiation, at genes related to neuronal lineage and regulation of membrane excitability. Consistent with these findings, the levels and activity of H3K9 methyltransferases (H3K9 HMT), but not H3K27 HMT, increased more prominently upon exposure to oligodendrocyte differentiating stimuli and were detected in stage-specific repressive protein complexes containing the transcription factors SOX10 or YY1. Silencing H3K9 HMT, but not H3K27 HMT, impaired oligodendrocyte differentiation and functionally altered the response of oligodendrocytes to electrical stimulation. Together, these results identify repressive H3K9 methylation as critical for gene repression during oligodendrocyte differentiation.

Identification of genes involved in the biology of atypical teratoid/rhabdoid tumours using Drosophila melanogaster.

Atypical teratoid/rhabdoid tumours (AT/RT) are malignant brain tumours. Unlike most other human brain tumours, AT/RT are characterized by inactivation of one single gene, SMARCB1. SMARCB1 is a member of the evolutionarily conserved SWI/SNF chromatin remodelling complex, which has an important role in the control of cell differentiation and proliferation. Little is known, however, about the pathways involved in the oncogenic effects of SMARCB1 inactivation, which might also represent targets for treatment. Here we report a comprehensive genetic screen in the fruit fly that revealed several genes not yet associated with loss of snr1, the Drosophila homologue of SMARCB1. We confirm the functional role of identified genes (including merlin, kibra and expanded, known to regulate hippo signalling pathway activity) in human rhabdoid tumour cell lines and AT/RT tumour samples. These results demonstrate that fly models can be employed for the identification of clinically relevant pathways in human cancer.

Transcript profiling of different types of multiple sclerosis lesions yields FGF1 as a promoter of remyelination.

Chronic demyelination is a pathological hallmark of multiple sclerosis (MS). Only a minority of MS lesions remyelinates completely. Enhancing remyelination is, therefore, a major aim of future MS therapies. Here we took a novel approach to identify factors that may inhibit or support endogenous remyelination in MS. We dissected remyelinated, demyelinated active, and demyelinated inactive white matter MS lesions, and compared transcript levels of myelination and inflammation-related genes using quantitative PCR on customized TaqMan Low Density Arrays. In remyelinated lesions, fibroblast growth factor (FGF) 1 was the most abundant of all analyzed myelination-regulating factors, showed a trend towards higher expression as compared to demyelinated lesions and was significantly higher than in control white matter. Two MS tissue blocks comprised lesions with adjacent de- and remyelinated areas and FGF1 expression was higher in the remyelinated rim compared to the demyelinated lesion core. In functional experiments, FGF1 accelerated developmental myelination in dissociated mixed cultures and promoted remyelination in slice cultures, whereas it decelerated differentiation of purified primary oligodendrocytes, suggesting that promotion of remyelination by FGF1 is based on an indirect mechanism. The analysis of human astrocyte responses to FGF1 by genome wide expression profiling showed that FGF1 induced the expression of the chemokine CXCL8 and leukemia inhibitory factor, two factors implicated in recruitment of oligodendrocytes and promotion of remyelination. Together, this study presents a transcript profiling of remyelinated MS lesions and identified FGF1 as a promoter of remyelination. Modulation of FGF family members might improve myelin repair in MS.