A functional role of NMDA receptor in regulating the differentiation of oligodendrocyte precursor cells and remyelination

Differentiation of oligodendrocyte precursor cells (OPCs) is the most important event for the myelination of central nervous system (CNS) axons during development and remyelination in demyelinating diseases, while the underlying molecular mechanisms remain largely unknown. Here we show that NMDA receptor (NMDAR) is a functional regulator of OPCs differentiation and remyelination. First, GluN1, GluN2A, and GluN2B subunits are expressed in oligodendrocyte lineage cells (OLs) in vitro and in vivo by immunostaining and Western blot analysis. Second, in a purified rat OPC culture system, NMDARs specially mediate OPCs differentiation by enhancing myelin proteins expression and the processes branching at the immature to mature oligodendrocyte transition analyzed by a serial of developmental stage‐specific antigens. Moreover, pharmacological NMDAR antagonists or specific knockdown of GluN1 by RNA interference in OPCs prevents the differentiation induced by NMDA. NMDA can activate the mammalian target of rapamycin (mTOR) signal in OPCs and the pro‐differentiation effect of NMDA is obstructed by the mTOR inhibitor rapamycin, suggesting NMDAR exerts its effect through mTOR‐dependent mechanism. Furthermore, NMDA increases numbers of myelin segments in DRG‐OPC cocultures. Finally, NMDAR specific antagonist MK801 delays remyelination in the cuprizone model examined by LFB‐PAS, immunofluorescence and electron microscopy. This effect appears to result from inhibiting OPCs differentiation as more NG2⁺ OPCs but less GST‐π⁺ mature oligodendrocytes are observed. Together, these results indicate that NMDAR plays a critical role in the regulation of OPCs differentiation in vitro and remyelination in cuprizone model which may provide potential target for the treatment of demyelination disease.     

Cyclin-dependent Kinase 18 Promotes Oligodendrocyte Precursor Cell Differentiation through Activating the Extracellular Signal-Regulated Kinase Signaling Pathway

The correct differentiation of oligodendrocyte precursor cells(OPCs) is essential for the myelination and remyelination processes in the central nervous system.Determining the regulatory mechanism is fundamental to the treatment of demyelinating diseases. By analyzing the RNA sequencing data of different neural cells, we found that cyclin-dependent kinase 18(CDK18) is exclusively expressed in oligodendrocytes. In vivo studies showed that the expression level of CDK18 gradually increased along with myelin formation during development and in the remyelination phase in a lysophosphatidylcholine-induced demyelination model, and was distinctively highly expressed in oligodendrocytes. In vitro overexpression and interference experiments revealed that CDK18 directly promotes the differentiation of OPCs, without affecting their proliferation or apoptosis. Mechanistically, CDK18 activated the RAS/mitogen-activated protein kinase kinase1/extracellular signal-regulated kinase pathway, thus promoting OPC differentiation. The results of the present study suggest that CDK18 is a promising cell-type specific target to treat demyelinating disease.     

SOX17 is expressed in regenerating oligodendrocytes in experimental models of demyelination and in multiple sclerosis

We have previously demonstrated that Sox17 expression is prominent at developmental stages corresponding to oligodendrocyte progenitor cell (OPC) cycle exit and onset of differentiation, and that Sox17 promotes initiation of OPC differentiation. In this study, we examined Sox17 expression and regulation under pathological conditions, particularly in two animal models of demyelination/remyelination and in post‐mortem multiple sclerosis (MS) brain lesions. We found that the number of Sox17 expressing cells was significantly increased in lysolecithin (LPC)‐induced lesions of the mouse spinal cord between 7 and 30 days post‐injection, as compared with controls. Sox17 immunoreactivity was predominantly detected in Olig2⁺ and CC1⁺ oligodendrocytes and rarely in NG2⁺ OPCs. The highest density of Sox17⁺ oligodendrocytes was observed at 2 weeks after LPC injection, coinciding with OPC differentiation. Consistent with these findings, in cuprizone‐treated mice, Sox17 expression was highest in newly generated and in maturing CC1⁺ oligodendrocytes, but low in NG2⁺ OPCs during the demyelination and remyelination phases. In MS tissue, Sox17 was primarily detected in actively demyelinating lesions and periplaque white matter. Sox17 immunoreactivity was co‐localized with NOGO‐A+ post‐mitotic oligodendrocytes both in active MS lesions and periplaque white matter. Taken together, our data: (i) demonstrate that Sox17 expression is highest in newly generated oligodendrocytes under pathological conditions and could be used as a marker of oligodendrocyte regeneration, and (ii) are suggestive of Sox17 playing a critical role in oligodendrocyte differentiation and lesion repair. GLIA 2013;61:1659–1672     

Pinocembrin Promotes OPC Differentiation and Remyelination via the mTOR Signaling Pathway

The exacerbation of progressive multiple sclerosis (MS) is closely associated with obstruction of the differentiation of oligodendrocyte progenitor cells (OPCs). To discover novel therapeutic compounds for enhancing remyelination by endogenous OPCs, we screened for myelin basic protein expression using cultured rat OPCs and a library of small-molecule compounds. One of the most effective drugs was pinocembrin, which remarkably promoted OPC differentiation and maturation without affecting cell proliferation and survival. Based on these in vitro effects, we further assessed the therapeutic effects of pinocembrin in animal models of demyelinating diseases. We demonstrated that pinocembrin significantly ameliorated the progression of experimental autoimmune encephalomyelitis (EAE) and enhanced the repair of demyelination in lysolectin-induced lesions. Further studies indicated that pinocembrin increased the phosphorylation level of mammalian target of rapamycin (mTOR). Taken together, our results demonstrated that pinocembrin promotes OPC differentiation and remyelination through the phosphorylated mTOR pathway, and suggest a novel therapeutic prospect for this natural flavonoid product in treating demyelinating diseases.     

UDP‐glucose enhances outward K+ currents necessary for cell differentiation and stimulates cell migration by activating the GPR17 receptor in oligodendrocyte precursors

In the developing and mature central nervous system, NG2 expressing cells comprise a population of cycling oligodendrocyte progenitor cells (OPCs) that differentiate into mature, myelinating oligodendrocytes (OLGs). OPCs are also characterized by high motility and respond to injury by migrating into the lesioned area to support remyelination. K⁺ currents in OPCs are developmentally regulated during differentiation. However, the mechanisms regulating these currents at different stages of oligodendrocyte lineage are poorly understood. Here we show that, in cultured primary OPCs, the purinergic G‐protein coupled receptor GPR17, that has recently emerged as a key player in oligodendrogliogenesis, crucially regulates K⁺ currents. Specifically, receptor stimulation by its agonist UDP‐glucose enhances delayed rectifier K⁺ currents without affecting transient K⁺ conductances. This effect was observed in a subpopulation of OPCs and immature pre‐OLGs whereas it was absent in mature OLGs, in line with GPR17 expression, that peaks at intermediate phases of oligodendrocyte differentiation and is thereafter downregulated to allow terminal maturation. The effect of UDP‐glucose on K⁺ currents is concentration‐dependent, blocked by the GPR17 antagonists MRS2179 and cangrelor, and sensitive to the K⁺ channel blocker tetraethyl‐ammonium, which also inhibits oligodendrocyte maturation. We propose that stimulation of K⁺ currents is responsible for GPR17‐induced oligodendrocyte differentiation. Moreover, we demonstrate, for the first time, that GPR17 activation stimulates OPC migration, suggesting an important role for this receptor after brain injury. Our data indicate that modulation of GPR17 may represent a strategy to potentiate the post‐traumatic response of OPCs under demyelinating conditions, such as multiple sclerosis, stroke, and brain trauma.     

Stage-dependent regulation of oligodendrocyte development and enhancement of myelin repair by dominant negative Master-mind 1 protein

Notch signaling has been implicated in the inhibition of oligodendrocyte differentiation and myelin gene expression during early development. However, inactivation of a particular Notch or Hes gene only produces a mild phenotype in oligodendrocyte development possibly due to the functional redundancies among closely related family members. To uncover the full role of Notch signaling in myelin development and regeneration, we generated the Sox10^rtTA/+; TetO-dnMAML1 double transgenic mice in which expression of dominant negative Master-mind 1 (dnMAML1) gene can be selectively induced in oligodendrocyte precursor cells (OPCs) for complete blockade of Notch signaling. It is found that dnMAML1 expression leads to robust precocious OL differentiation and premature axonal myelination in the spinal cord, possibly by upregulating Nkx2.2 and downregulating Pdgfra expression. Unexpectedly, at late embryonic stages, dnMAML1 expression dramatically increased the number of OPCs, indicating a stage-dependent effect of Notch signaling on OPC proliferation. In addition, dnMAML1 also significantly enhances axonal remyelination following chemical-induced demyelination, providing a promising therapeutic target for lesion repair in demyelinating disease.     

The fatty acid binding protein FABP7 is required for optimal oligodendrocyte differentiation during myelination but not during remyelination

The major constituents of the myelin sheath are lipids, which are made up of fatty acids (FAs). The hydrophilic environment inside the cells requires FAs to be bound to proteins, preventing their aggregation. Fatty acid binding proteins (FABPs) are one class of proteins known to bind FAs in a cell. Given the crucial role of FAs for myelin sheath formation we investigated the role of FABP7, the major isoform expressed in oligodendrocyte progenitor cells (OPCs), in developmental myelination and remyelination. Here, we show that the knockdown of Fabp7 resulted in a reduction of OPC differentiation in vitro. Consistent with this result, a delay in developmental myelination was observed in Fabp7 knockout animals. This delay was transient with full myelination being established before adulthood. FABP7 was dispensable for remyelination, as the knockout of Fapb7 did not alter remyelination efficiency in a focal demyelination model. In summary, while FABP7 is important in OPC differentiation in vitro, its function is not crucial for myelination and remyelination in vivo.     

Diosgenin promotes oligodendrocyte progenitor cell differentiation through estrogen receptor-mediated ERK1/2 activation to accelerate remyelination

Differentiation of oligodendrocyte progenitor cells (OPCs) into mature oligodendrocytes is a prerequisite for remyelination after demyelination, and impairment of this process is suggested to be a major reason for remyelination failure. Diosgenin, a plant-derived steroid, has been implicated for therapeutic use in many diseases, but little is known about its effect on the central nervous system. In this study, using a purified rat OPC culture model, we show that diosgenin significantly and specifically promotes OPC differentiation without affecting the viability, proliferation, or migration of OPC. Interestingly, the effect of diosgenin can be blocked by estrogen receptor (ER) antagonist ICI 182780 but not by glucocorticoid and progesterone receptor antagonist RU38486, nor by mineralocorticoid receptor antagonist spirolactone. Moreover, it is revealed that both ER-alpha and ER-beta are expressed in OPC, and diosgenin can activate the extracellular signal-regulated kinase 1/2 (ERK1/2) in OPC via ER. The pro-differentiation effect of diosgenin can also be obstructed by the ERK inhibitor PD98059. Furthermore, in the cuprizone-induced demyelination model, it is demonstrated that diosgenin administration significantly accelerates/enhances remyelination as detected by Luxol fast blue stain, MBP immunohistochemistry and real time RT-PCR. Diosgenin also increases the number of mature oligodendrocytes in the corpus callosum while it does not affect the number of OPCs. Taking together, our results suggest that diosgenin promotes the differentiation of OPC into mature oligodendrocyte through an ER-mediated ERK1/2 activation pathway to accelerate remyelination, which implicates a novel therapeutic usage of this steroidal natural product in demyelinating diseases such as multiple sclerosis (MS).     

Shikimic Acid Promotes Oligodendrocyte Precursor Cell Differentiation and Accelerates Remyelination in Mice

The obstacle to successful remyelination in demyelinating diseases, such as multiple sclerosis, mainly lies in the inability of oligodendrocyte precursor cells(OPCs) to differentiate, since OPCs and oligodendrocytelineage cells that are unable to fully differentiate are found in the areas of demyelination. Thus, promoting the differentiation of OPCs is vital for the treatment of demyelinating diseases. Shikimic acid(SA) is mainly derived from star anise, and is reported to have antiinfluenza, anti-oxidation, and anti-tumor effects. In the present study, we found that SA significantly promoted the differentiation of cultured rat OPCs without affecting their proliferation and apoptosis. In mice, SA exerted therapeutic effects on experimental autoimmune encephalomyelitis(EAE), such as alleviating clinical EAE scores, inhibiting inflammation, and reducing demyelination in the CNS. SA also promoted the differentiation of OPCs as well as their remyelination after lysolecithin-induced demyelination.Furthermore, we showed that the promotion effect of SA on OPC differentiation was associated with the up-regulation of phosphorylated m TOR. Taken together, our resultsdemonstrated that SA could act as a potential drug candidate for the treatment of demyelinating diseases.     

Oligodendroglial lineage cells express nuclear p57kip2 in multiple sclerosis lesions

Impaired remyelination in multiple sclerosis (MS) might be due to the failure of oligodendrocyte precursor cells (OPC) to differentiate into myelinating oligodendrocytes. Animal experimental data have shown that p57kip2 inhibits oligodendroglial differentiation, indicating that this factor could contribute to remyelination failure. This study investigates oligodendroglial p57kip2 expression and its association with remyelination in MS lesions. To analyze the potential association of p57kip2 expression with human oligodendroglial maturation, double immunofluorescence staining was performed on brain tissue from 30 MS patients and 20 controls. Anti‐p57kip2 antibody was combined with either anti‐Nogo‐A to label mature oligodendrocytes or anti‐Olig2 antibodies to identify immature OPCs. We evaluated MS lesions with or without remyelination, the periplaque white matter (PPWM) as well as control white matter (WM). p57kip2‐expressing cells were assessed and correlated with the extent of remyelination. Most Nogo‐A‐positive oligodendrocytes (range, 87–98%) and all Olig2^strong‐positive OPCs expressed p57kip2 in MS lesions, in the PPWM and in control WM. p57kip2 expression in oligodendrocytes and OPCs were similar in MS lesions with remyelination compared to MS lesions lacking remyelination. Interestingly, all oligodendroglial lineage cells showed nuclear p57kip2 expression only, with mature oligodendrocytes expressing p57kip2 at low or intermediate levels and OPCs featuring strong expression levels, indicating that this factor may be dynamically expressed during maturation processes. Therefore, p57kip2 appears to be widely expressed in the human oligodendroglial lineage, and potential beneficial effects on remyelination in the MS brain are not based on subcellular p57kip2 localization shifts, as suggested by previous animal experiments. GLIA 2013;61:1250–1260     

Histone deacetylase activity is required for human oligodendrocyte progenitor differentiation

The molecular mechanisms controlling human oligodendrocyte development are poorly characterized. Microarray analysis of human oligodendrocyte progenitor cells (OPCs) and immature oligodendrocytes revealed that specific‐class I histone deacetylase (HDAC) target genes were actively repressed during oligodendrocyte commitment. Although epigenetic regulation of oligodendrocyte differentiation has been established in rodent development, the role of HDACs in human OPCs remains undefined. We used HDAC inhibitors (HDACi) trichostatin A (TSA) and sodium butyrate to determine the importance of HDAC activity in human primary OPC differentiation. Treatment with either drug resulted in significant dose‐dependent inhibition of O4⁺ oligodendrocyte cell differentiation, reduction of oligodendrocyte morphological maturation, and downregulation of myelin basic protein mRNA. High dose TSA treatment was also associated with reduction in OPC proliferation. HDACi treatment prevented downregulation of SOX2, ID4, and TCF7L2 mRNAs but did not regulate HES5, suggesting that targets of HDAC repression may differ between species. These results predict that HDACi treatment would impair proliferation and differentiation by parenchymal oligodendrocyte progenitors, and thereby degrade their potential for endogenous repair in human demyelinating disease. © 2012 Wiley Periodicals, Inc.     

Osteopontin is extensively expressed by macrophages following CNS demyelination but has a redundant role in remyelination

Osteopontin (OPN) is a key immunoregulator in the autoimmune-mediated demyelinating disease multiple sclerosis. OPN may also play a role in the remyelination since it is 1) a ligand for αV integrins, several of which regulate the properties of the oligodendrocyte precursor cells (OPCs) primarily responsible for remyelination, and 2) enhances myelin membrane formation in OPC lines. Here we show that OPN is expressed at high levels during remyelination of toxin-induced demyelination. The increased expression is due to mRNA expression in macrophages and follows differences in macrophage responses to demyelination in young and old adult animals. To identify the role of OPN in remyelination focal demyelination was induced in wild-type and OPN^−/− mice. There was no difference in the rate of remyelination between the two groups indicating that OPN is not a critical component of remyelination.     

The presence of astrocytes in areas of demyelination influences remyelination following transplantation of oligodendrocyte progenitors

To date, most experiments examining the myelination potential of transplanted cells have been undertaken into either the immature nervous system or into acutely demyelinating lesions. Since these are situations where myelination or remyelination are occurring, such studies provide little information on the likely outcome of introducing myelinogenic cells into area of chronic demyelination. In an attempt to gain a greater understanding of the interaction between astrocytes and oligodendrocyte progenitors in areas of demyelination, we undertook transplantation experiments in which an identical preparation of oligodendrocyte progenitors (OPCs) was (1) transplanted directly into astrocyte-free areas of acute demyelination (3 days after induction), (2) transplanted cranial to similar areas of demyelination (20 days after induction) or (3) transplanted cranial to areas of demyelination (20 days after induction) that had been injected with astrocytes at 3 days to confront OPCs with demyelinated axons in an astrocytic environment. The acute astrocyte-free lesions were remyelinated by oligodendrocytes and Schwann cells while the delayed interaction of OPCs with demyelinating lesions resulted in only oligodendrocyte remyelination, the extent of which was reduced when the area of demyelination contained astrocytes. The results of these experiments illustrate that the introduction of OPCs into an astrocyte-free area of demyelination soon after its induction favours Schwann cell differentiation while the presence of established astrocytes in an area of demyelination has an inhibitory effect on the extent of oligodendrocyte remyelination achieved by OPCs.     

mTOR Signaling Regulates Metabolic Function in Oligodendrocyte Precursor Cells and Promotes Efficient Brain Remyelination in the Cuprizone Model

In demyelinating diseases, such as multiple sclerosis, primary loss of myelin and subsequent neuronal degeneration throughout the CNS impair patient functionality. While the importance of mechanistic target of rapamycin (mTOR) signaling during developmental myelination is known, no studies have yet directly examined the function of mTOR signaling specifically in the oligodendrocyte (OL) lineage during remyelination. Here, we conditionally deleted Mtor from adult oligodendrocyte precursor cells (OPCs) using Ng2-Cre^ERT in male adult mice to test its function in new OLs responsible for remyelination. During early remyelination after cuprizone-induced demyelination, mice lacking mTOR in adult OPCs had unchanged OL numbers but thinner myelin. Myelin thickness recovered by late-stage repair, suggesting a delay in myelin production when Mtor is deleted from adult OPCs. Surprisingly, loss of mTOR in OPCs had no effect on efficiency of remyelination after lysophosphatidylcholine lesions in either the spinal cord or corpus callosum, suggesting that mTOR signaling functions specifically in a pathway dysregulated by cuprizone to promote remyelination efficiency. We further determined that cuprizone and inhibition of mTOR cooperatively compromise metabolic function in primary rat OLs undergoing differentiation. Together, our results support the conclusion that mTOR signaling in OPCs is required to overcome the metabolic dysfunction in the cuprizone-demyelinated adult brain.SIGNIFICANCE STATEMENT Impaired remyelination by oligodendrocytes contributes to the progressive pathology in multiple sclerosis, so it is critical to identify mechanisms of improving remyelination. The goal of this study was to examine mechanistic target of rapamycin (mTOR) signaling in remyelination. Here, we provide evidence that mTOR signaling promotes efficient remyelination of the brain after cuprizone-mediated demyelination but has no effect on remyelination after lysophosphatidylcholine demyelination in the spinal cord or brain. We also present novel data revealing that mTOR inhibition and cuprizone treatment additively affect the metabolic profile of differentiating oligodendrocytes, supporting a mechanism for the observed remyelination delay. These data suggest that altered metabolic function may underlie failure of remyelination in multiple sclerosis lesions and that mTOR signaling may be of therapeutic potential for promoting remyelination.