Regulates Oligodendrocyte Differentiation Research Articles

Regulation of Oligodendrocyte Differentiation and Myelination

Despite the importance of myelin for the rapid conduction of action potentials, the molecular bases of oligodendrocyte differentiation and central nervous system (CNS) myelination are still incompletely understood. Recent results have greatly advanced this understanding, identifying new transcriptional regulators of myelin gene expression, elucidating vital roles for microRNAs in controlling myelination, and clarifying the extracellular signaling mechanisms that orchestrate the development of myelin. Studies have also demonstrated an unexpected level of plasticity of myelin in the adult CNS. These recent advances provide new insight into how remyelination may be stimulated in demyelinating disorders such as multiple sclerosis.

The QKI-6 RNA Binding Protein Regulates Actin-interacting Protein-1 mRNA Stability during Oligodendrocyte Differentiation

The quaking viable (qk(v)) mice represent an animal model of dysmyelination. The absence of expression of the QKI-6 and QKI-7 cytoplasmic isoforms in oligodendrocytes (OLs) during CNS myelination causes the qk(v) mouse phenotype. The QKI RNA-binding proteins are known to regulate RNA metabolism of cell cycle proteins and myelin components in OLs; however, little is known of their role in reorganizing the cytoskeleton or process outgrowth during OL maturation and differentiation. Here, we identify the actin-interacting protein (AIP)-1 mRNA as a target of QKI-6 by using two-dimensional differential gel electrophoresis. The AIP-1 mRNA contains a consensus QKI response element within its 3'-untranslated region that, when bound by QKI-6, decreases the half-life of the AIP-1 mRNA. Although the expression of QKI-6 is known to increase during OL differentiation and CNS myelination, we show that this increase is paralleled with a corresponding decrease in AIP-1 expression in rat brains. Furthermore, qk(v)/qk(v) mice that lack QKI-6 and QKI-7 within its OLs had an increased level of AIP-1 in OLs. Moreover, primary rat OL precursors harboring an AIP-1 small interfering RNA display defects in OL process outgrowth. Our findings suggest that the QKI RNA-binding proteins regulate OL differentiation by modulating the expression of AIP-1.

Mitogen‐activated protein kinase activated protein kinase 2 (MK2) participates in p38 MAPK regulated control of oligodendrocyte differentiation

The p38 mitogen-activated protein kinases (p38 MAPKs) are a family of kinases that regulate a number of cellular functions including cell migration, proliferation, and differentiation. We have previously reported a role for p38 MAPK in the regulation of oligodendrocyte (OLG) differentiation and Schwann cell myelination. Here, we extend our previous findings by showing that a p38 substrate, mitogen-activated protein kinase activated protein kinase 2 (MK2) is a downstream element of the p38 signaling pathway responsible for effecting OLG differentiation. Inhibition of MK2 activity in oligodendrocyte progenitors (OLPs) using CMPD1 [4-(2'-fluorobiphenyl-4-yl)-N-(4-hydroxyphenyl)-butyramide] blocked the activation of MK2 and resulted in decreased accumulation of myelin-differentiation markers, including myelin-associated glycoprotein (MAG) and myelin basic protein (MBP). We corroborated these findings using a small-interfering RNA to MK2, which decreased the myelin-specific lipid galactosylceramide and MAG. Treatment of cultures with CMPD1 decreased the steady state levels of mRNA encoding myelin transcription factor 1 (Myt1), MAG, MBP, and Opalin, a transmembrane sialylglycoprotein expressed in oligodendrocytes. In contrast, increases were observed in the mRNA levels of OLG transcriptional repressors, including transcription factor 4 (Tcf4), Notch1, and inhibitor of differentiation 2 (Id2). Furthermore, we found that the predominantly expressed isoform of p38 in OLGs, p38alpha, and MK2 can form coimmunoprecipitable complexes in OLPs and OLGs. Our results demonstrate that the p38-MK2 pathway is a component of the signaling cascade regulating OLG differentiation.

Tyrosine phosphatases Shp1 and Shp2 have unique and opposing roles in oligodendrocyte development

Oligodendrocyte progenitor cells first proliferate to generate sufficient cell numbers and then differentiate into myelin-producing oligodendrocytes. The signal transduction mediators that underlie these events, however, remain poorly understood. The tyrosine phosphatase Shp1 has been linked to oligodendrocyte differentiation as Shp1-deficient mice show hypomyelination. The Shp1 homolog, Shp2, has recently been shown to regulate astrogliogenesis, but its role in oligodendrocyte development remains unknown. Here, we report that Shp2 protein levels were developmentally regulated in oligodendrocytes, with Shp2 phosphorylation being promoted by oligodendroglial mitogens but suppressed by laminin, an extracellular matrix protein that promotes oligodendroglial differentiation. In contrast, oligodendrocyte progenitors were found to be unresponsive to mitogens following Shp2, but not Shp1, depletion. In agreement with previous studies, Shp1 depletion led to decreased levels of myelin basic protein in differentiating oligodendrocytes, as well as reduced outgrowth of myelin membrane sheets. Shp2 depletion in contrast did not prevent oligodendrocyte differentiation but promoted expanded myelin membrane outgrowth. Taken together these data suggest that Shp1 and Shp2 have distinct functions in oligodendrocyte development: Shp2 regulates oligodendrocyte progenitor proliferation and Shp1 regulates oligodendrocyte differentiation. Adhesion to laminin may additionally provide extrinsic regulation of Shp2 activity and thus promote the transition from progenitor to differentiating oligodendrocyte.

Genome-wide Association Study in a High-Risk Isolate for Multiple Sclerosis Reveals Associated Variants in STAT3 Gene

Genetic risk for multiple sclerosis (MS) is thought to involve both common and rare risk alleles. Recent GWAS and subsequent meta-analysis have established the critical role of the HLA locus and identified new common variants associated to MS. These variants have small odds ratios (ORs) and explain only a fraction of the genetic risk. To expose potentially rare, high-impact alleles, we conducted a GWAS of 68 distantly related cases and 136 controls from a high-risk internal isolate of Finland with increased prevalence and familial occurrence of MS. The top 27 loci with p < 10(-4) were tested in 711 cases and 1029 controls from Finland, and the top two findings were validated in 3859 cases and 9110 controls from more heterogeneous populations. SNP (rs744166) within the STAT3 gene was associated to MS (p = 2.75 x 10(-10), OR 0.87, confidence interval 0.83-0.91). The protective haplotype for MS in STAT3 is a risk allele for Crohn disease, implying that STAT3 represents a shared risk locus for at least two autoimmune diseases. This study also demonstrates the potential of special isolated populations in search for variants contributing to complex traits.

Notch1 signaling plays a role in regulating precursor differentiation during CNS remyelination

In the developing CNS, Notch1 and its ligand, Jagged1, regulate oligodendrocyte differentiation and myelin formation, but their role in repair of demyelinating lesions in diseases such as multiple sclerosis remains unresolved. To address this question, we generated a mouse model in which we targeted Notch1 inactivation to oligodendrocyte progenitor cells (OPCs) using Olig1Cre and a floxed Notch1 allele, Notch1(12f). During CNS development, OPC differentiation was potentiated in Olig1Cre:Notch1(12f/12f) mice. Importantly, in adults, remyelination of demyelinating lesions was also accelerated, at the expense of proliferation within the progenitor population. Experiments in vitro confirmed that Notch1 signaling was permissive for OPC expansion but inhibited differentiation and myelin formation. These studies also revealed that astrocytes exposed to TGF-beta1 restricted OPC maturation via Jagged1-Notch1 signaling. These data suggest that Notch1 signaling is one of the mechanisms regulating OPC differentiation during CNS remyelination. Thus, Notch1 may represent a potential therapeutical avenue for lesion repair in demyelinating disease.

HDAC1 and HDAC2 regulate oligodendrocyte differentiation by disrupting the β-catenin–TCF interaction

Oligodendrocyte development is regulated by the interplay of repressors and activators in a complex transcriptional network. Here we report that two histone-modifying enzymes, HDAC1 and HDAC2, are required for oligodendrocyte formation. Genetic deletion of both HDAC1 and HDAC2 in oligodendrocyte lineage cells resulted in stabilization and nuclear translocation of β-catenin, which negatively regulates oligodendrocyte development by repressing Olig2 expression. We further identified an oligodendrocyte-restricted transcription factor TCF7L2/TCF4 as a bipartite co-effector of β-catenin for regulating oligodendrocyte differentiation. Targeted disruption of TCF7L2 in mice leads to severe defects in oligodendrocyte maturation, while expression of its dominant repressive form promotes precocious oligodendrocyte specification in developing chick neural tube. Transcriptional co-repressors HDAC1 and HDAC2 compete with β-catenin for TCF7L2 interaction to regulate downstream genes involved in oligodendrocyte differentiation. Hence, crosstalk between HDAC1/2 and the canonical Wnt signaling pathway mediated by TCF7L2 serves as a regulatory mechanism for oligodendrocyte differentiation.

LINGO-1 Interacts with WNK1 to Regulate Nogo-induced Inhibition of Neurite Extension

LINGO-1 is a component of the tripartite receptor complexes, which act as a convergent mediator of the intracellular signaling in response to myelin-associated inhibitors and lead to collapse of growth cone and inhibition of neurite extension. Although the function of LINGO-1 has been intensively studied, its downstream signaling remains elusive. In the present study, a novel interaction between LINGO-1 and a serine-threonine kinase WNK1 was identified by yeast two-hybrid screen. The interaction was further validated by fluorescence resonance energy transfer and co-immunoprecipitation, and this interaction was intensified by Nogo66 treatment. Morphological evidences showed that WNK1 and LINGO-1 were co-localized in cortical neurons. Furthermore, either suppressing WNK1 expression by RNA interference or overexpression of WNK1-(123-510) attenuated Nogo66-induced inhibition of neurite extension and inhibited the activation of RhoA. Moreover, WNK1 was identified to interact with Rho-GDI1, and this interaction was attenuated by Nogo66 treatment, further indicating its regulatory effect on RhoA activation. Taken together, our results suggest that WNK1 is a novel signaling molecule involved in regulation of LINGO-1 mediated inhibition of neurite extension.

Activation of the Mammalian Target of Rapamycin (mTOR) Is Essential for Oligodendrocyte Differentiation

Although both extrinsic and intrinsic factors have been identified that orchestrate the differentiation and maturation of oligodendrocytes, less is known about the intracellular signaling pathways that control the overall commitment to differentiate. Here, we provide evidence that activation of the mammalian target of rapamycin (mTOR) is essential for oligodendrocyte differentiation. Specifically, mTOR regulates oligodendrocyte differentiation at the late progenitor to immature oligodendrocyte transition as assessed by the expression of stage specific antigens and myelin proteins including MBP and PLP. Furthermore, phosphorylation of mTOR on Ser 2448 correlates with myelination in the subcortical white matter of the developing brain. We demonstrate that mTOR exerts its effects on oligodendrocyte differentiation through two distinct signaling complexes, mTORC1 and mTORC2, defined by the presence of the adaptor proteins raptor and rictor, respectively. Disrupting mTOR complex formation via siRNA mediated knockdown of raptor or rictor significantly reduced myelin protein expression in vitro. However, mTORC2 alone controlled myelin gene expression at the mRNA level, whereas mTORC1 influenced MBP expression via an alternative mechanism. In addition, investigation of mTORC1 and mTORC2 targets revealed differential phosphorylation during oligodendrocyte differentiation. In OPC-DRG cocultures, inhibiting mTOR potently abrogated oligodendrocyte differentiation and reduced numbers of myelin segments. These data support the hypothesis that mTOR regulates commitment to oligodendrocyte differentiation before myelination.

Loss of Fyn tyrosine kinase on the C57BL/6 genetic background causes hydrocephalus with defects in oligodendrocyte development

The supportive functions of oligodendrocytes are required for the survival and development of axons, ensuring the organization of highly specialized neuronal networks in brain. Although the molecules that regulate oligodendrocyte differentiation in vitro have been identified, their roles in vivo are largely uncertain. Here we report that fyn deficiency on the C57BL/6 genetic background resulted in premature death, showing severe hydrocephalus with neonatal onset. One week after birth, fyn-deficient mice showed enlarged lateral ventricles with thinner cerebral cortices and degenerating axons in the corpus callosum. In addition, before the onset of myelination, the number of oligodendrocytes was reduced and their morphogenesis was impaired in the cerebral cortex. These results demonstrate that Fyn is essential for normal brain development and suggest that defects in oligodendrocyte development cause degeneration of cortical axons and subsequent hydrocephalus in fyn-deficient mice.

SOX5 Controls the Sequential Generation of Distinct Corticofugal Neuron Subtypes

The molecular mechanisms controlling the development of distinct subtypes of neocortical projection neurons, and CNS neuronal diversity more broadly, are only now emerging. We report that the transcription factor SOX5 controls the sequential generation of distinct corticofugal neuron subtypes by preventing premature emergence of normally later-born corticofugal neurons. SOX5 loss-of-function causes striking overlap of the identities of the three principal sequentially born corticofugal neuron subtypes: subplate neurons, corticothalamic neurons, and subcerebral projection neurons. In Sox5(-/-) cortex, subplate neurons aberrantly develop molecular hallmarks and connectivity of subcerebral projection neurons; corticothalamic neurons are imprecisely differentiated, while differentiation of subcerebral projection neurons is accelerated. Gain-of-function analysis reinforces the critical role of SOX5 in controlling the sequential generation of corticofugal neurons--SOX5 overexpression at late stages of corticogenesis causes re-emergence of neurons with corticofugal features. These data indicate that SOX5 controls the timing of critical fate decisions during corticofugal neuron production and thus subtype-specific differentiation and neocortical neuron diversity.

P38 mitogen‐activated protein kinase is required for central nervous system myelination

The p38 MAPKs are a family of kinases that regulate a number of cellular functions including cell migration, proliferation, and differentiation. Here, we report that p38 regulates oligodendrocyte differentiation. Inhibition of p38 with PD169316 and SB203580 prevented accumulation of protein and mRNA of cell-stage specific markers characteristic of differentiated oligodendrocytes, including myelin basic protein, myelin-associated glycoprotein, and the glycosphingolipids, galactosylceramide and sulfatide. In addition, the cell cycle regulator p27(kip1) and the transcription factor Sox10 were also significantly reduced. Most significantly, p38 inhibitors completely and irreversibly blocked myelination of dorsal root ganglion neurons by oligodendrocytes and prevented the axolemmal organization of the axo-glial adhesion molecule Caspr. Our results suggest a role(s) for this kinase in key regulatory steps in the maturation of OLGs and initiation of myelination.

Surprising synapses deep in the brain

Two independent groups in this issue report synaptic communication between callosal axons and the NG2-positive glial precursors in the brain. Activity-dependent vesicular glutamate release from axon collaterals triggers AMPA-type currents in the precursors. This phenomenon might be involved in regulation of oligodendrocyte differentiation or remyelination.

P38 MAP Kinase Regulation of Oligodendrocyte Differentiation with CREB as a Potential Target

Despite a substantial understanding of the factors regulating oligodendrocyte differentiation, the signaling mechanisms involved in this process are not well-understood. This study elaborates on the findings (Bhat NR, Zhang P (1997) FASEB J 11:A925; Baron W, Metz B, Bansal R, Hoekstra D, de Vries H (2000) Mol Cell Neurosci 15:314-329) of a role for p38 MAP kinase signaling in oligodendrocyte differentiation and myelin gene expression. When proliferating oligodendrocyte progenitors were switched to a growth factor-free differentiation medium, there was a rapid activation of p38 kinase that correlated with an increased phosphorylation of CREB, a down-stream target and a factor involved in oligodendrocyte differentiation. Addition of forskolin, a known inducer of intracellular c-AMP and of oligodendrocyte differentiation, also stimulated CREB phosphorylation in a p38 kinase dependent way. Pharmacological inhibition of p38 interfered with the morphological and antigenic changes associated with differentiating oligodendrocytes as well as with the developmental and forskolin-induced expression of myelin basic protein, thereby supporting an essential role for p38 MAPK pathway in oligodendrocyte differentiation.

The Structure of the Lingo-1 Ectodomain, a Module Implicated in Central Nervous System Repair Inhibition

Nogo receptor (NgR)-mediated control of axon growth relies on the central nervous system-specific type I transmembrane protein Lingo-1. Interactions between Lingo-1 and NgR, along with a complementary co-receptor, result in neurite and axonal collapse. In addition, the inhibitory role of Lingo-1 is particularly important in regulation of oligodendrocyte differentiation and myelination, suggesting that pharmacological modulation of Lingo-1 function could be a novel approach for nerve repair and remyelination therapies. Here we report on the crystal structure of the ligand-binding ectodomain of human Lingo-1 and show it has a bimodular, kinked structure composed of leucine-rich repeat (LRR) and immunoglobulin (Ig)-like modules. The structure, together with biophysical analysis of its solution properties, reveals that in the crystals and in solution Lingo-1 persistently associates with itself to form a stable tetramer and that it is its LRR-Ig-composite fold that drives such assembly. Specifically, in the crystal structure protomers of Lingo-1 associate in a ring-shaped tetramer, with each LRR domain filling an open cleft in an adjacent protomer. The tetramer buries a large surface area (9,200 A2) and may serve as an efficient scaffold to simultaneously bind and assemble the NgR complex components during activation on a membrane. Potential functional binding sites that can be identified on the ectodomain surface, including the site of self-recognition, suggest a model for protein assembly on the membrane.

Human QKI, a potential regulator of mRNA expression of human oligodendrocyte-related genes involved in schizophrenia

The quaking viable mouse mutation (qk(v)) is a deletion including the 5' regulatory region of the quaking gene (Qki), which causes body tremor and severe dysmyelination in mouse. The function of the human quaking gene, called quaking homolog KH domain RNA-binding (mouse) (QKI), is not well known. We have previously shown that QKI is a new candidate gene for schizophrenia. Here we show that human QKI mRNA levels can account for a high proportion (47%) of normal interindividual mRNA expression variation (and covariation) of six oligodendrocyte-related genes (PLP1, MAG, MBP, TF, SOX10, and CDKN1B) in 55 human brain autopsy samples from individuals without psychiatric diagnoses. In addition, the tightly coexpressed myelin-related genes (PLP1, MAG, and TF) have decreased mRNA levels in 55 schizophrenic patients, as compared with 55 control individuals, and most of this difference (68-96%) can be explained by variation in the relative mRNA levels of QKI-7kb, the same QKI splice variant previously shown to be down-regulated in patients with schizophrenia. Taken together, our results suggest that QKI levels may regulate oligodendrocyte differentiation and maturation in human brain, in a similar way as in mouse. Moreover, we hypothesize that previously observed decreased activity of myelin-related genes in schizophrenia might be caused by disturbed QKI splicing.

Advancement of differentiation of oligodendrocyte progenitor cells by a cascade including protein kinase A and cyclic AMP-response element binding protein

A transcription factor, cyclic AMP-response element binding protein (CREB), which is phosphorylated by protein kinases (PKA and PKC), is known to be involved in the regulation of oligodendrocyte differentiation. However, it is still unclear whether protein kinase A (PKA) and protein kinase C (PKC) are used simultaneously or at different time points to phosphorylate CREB in oligodendrocytes and whether CREB phosphorylation advances oligodendrocyte differentiation or vise versa. Our previous experiments have shown that in the differentiation process from immature to mature cells, CREB phosphorylation depends on PKC activity and leads to the progression of differentiation. In order to gain a better understanding of the process of differentiation from progenitor to immature cells, we identified which protein kinase, i.e., PKA or PKC, regulates CREB phosphorylation and we determined whether CREB phosphorylation advances differentiation or the reverse. Our results showed that CREB phosphorylation is principally regulated by PKA activity in progenitor cells but not by PKC activity, and that this phosphorylation advances the differentiation of progenitor cells to immature cells in oligodendrocytes.

Differentiation of immature oligodendrocytes is regulated by phosphorylation of cyclic AMP-response element binding protein by a protein kinase C signaling cascade

Previous experiments showed that the expression and phosphorylation levels of cyclic AMP-response element binding protein (CREB) are important factors that regulate oligodendrocyte differentiation. The present study was designed to determine whether CREB phosphorylation advances oligodendrocyte differentiation or vice versa and to identify the protein kinase that primarily regulates CREB phosphorylation. We examined the expression and phosphorylation levels of CREB in developing oligodendrocytes at a specific differentiation stage by double-immunocytochemical staining with specific differentiation markers and antibody for phosphorylated CREB. We found that the CREB expression level increased along oligodendrocyte differentiation, and that its phosphorylated level was highest in immature oligodendrocytes. We also showed that CREB phosphorylation was regulated principally by protein kinase C (PKC) activity in immature oligodendrocytes. Our findings suggest that CREB phosphorylation is dependent on a PKC signaling cascade, and this phosphorylation activates CREB-mediated transcription and advances the differentiation of immature to mature oligodendrocytes.

Sigma-1 receptors at galactosylceramide-enriched lipid microdomains regulate oligodendrocyte differentiation.

In the brain, myelin is important in regulating nerve conduction and neurotransmitter release by providing insulation at axons. Myelin is a specialized yet continuous sheet structure of differentiated oligodendrocytes (OLs) that is enriched in lipids, specifically galactosylceramides (GalCer) originated at the endoplasmic reticulum (ER). GalCer are known to affect OL differentiation. However, the mechanism whereby GalCer affect OL differentiation is not well understood. Sigma-1 receptors (Sig-1Rs), shown by us to exist in detergent-insoluble lipid microdomains at lipid-enriched loci of ER in NG108 cells, are important in the compartmentalization/transport of ER-synthesized lipids and in cellular differentiation. In this study, we used rat primary hippocampal cultures and found that Sig-1Rs form GalCer-enriched lipid rafts at ER lipid droplet-like structures in the entire myelin sheet of mature OLs. In rat OL progenitors (CG-4 cells), levels of lipid raft-residing Sig-1Rs and GalCer increase as cells differentiate. Sig-1Rs also increase in OLs and myelin of developing rat brains. Sig-1R, GalCer, and cholesterol are colocalized and are resistant to the Triton X-100 solubilization. Treating cells with a Sig-1R agonist or targeting Sig-1Rs at lipid rafts by overexpression of Sig-1Rs in CG-4 cells enhances differentiation, whereas reducing Sig-1Rs at lipid rafts by transfection of functionally dominant-negative Sig-1Rs attenuates differentiation. Furthermore, Sig-1R siRNA inhibits differentiation. Our findings indicate that, in the brain, Sig-1Rs targeting GalCer-containing lipid microdomains are important for OL differentiation and that Sig-1Rs may play an important role in the pathogenesis of certain demyelinating diseases.

Tyrosine phosphorylation of p190 RhoGAP by Fyn regulates oligodendrocyte differentiation.

During development of the central nervous system, oligodendrocyte progenitor cells differentiate into mature myelinating cells. The molecular signals that promote this process, however, are not well defined. One molecule that has been implicated in oligodendrocyte differentiation is the Src family kinase Fyn. In order to probe the function of Fyn in this system, a yeast two hybrid screen was performed. Using Fyn as bait, p190 RhoGAP was isolated in the screen of an oligodendrocyte cDNA library. Coimmunoprecipitation and in vitro binding assays verified that p190 RhoGAP bound to the Fyn SH2 domain. Phosphorylation of p190 required active Fyn tyrosine kinase and was increased threefold upon differentiation of primary oligodendrocytes. Moreover, complex formation between p190 and p120 RasGAP occurred in differentiated oligodendrocytes. p190 RhoGAP activity is known to regulate the RhoGDP:RhoGTP ratio. Indeed, expression of dominant negative Rho in primary oligodendrocytes caused a hyperextension of processes. Conversely, constitutively activated Rho caused reduced process formation. These findings define a pathway in which Fyn activity regulates the phosphorylation of p190, leading to an increase in RhoGAP activity with a subsequent increase in RhoGDP, which in turn, regulates the morphological changes that accompany oligodendrocyte differentiation.