Distinct age and differentiation-state dependent metabolic profiles of oligodendrocytes under optimal and stress conditions.

Within the microenvironment of multiple sclerosis lesions, oligodendrocytes are subject to metabolic stress reflecting effects of focal ischemia and inflammation. Previous studies have shown that under optimal conditions in vitro, the respiratory activity of human adult brain-derived oligodendrocytes is lower and more predominantly glycolytic compared to oligodendrocytes differentiated in vitro from post natal rat brain oligodendrocyte progenitor cells. In response to sub-lethal metabolic stress, adult human oligodendrocytes reduce overall energy production rate impacting the capacity to maintain myelination. Here, we directly compare the metabolic profiles of oligodendrocytes derived from adult rat brain with oligodendrocytes newly differentiated in vitro from oligodendrocyte progenitor cells obtained from the post natal rat brain, under both optimal culture and metabolic stress (low/no glucose) conditions. Oxygen consumption and extracellular acidification rates were measured using a Seahorse extracellular flux analyzer. Our findings indicate that under optimal conditions, adult rat oligodendrocytes preferentially use glycolysis whereas newly differentiated post natal rat oligodendrocytes, and the oligodendrocyte progenitor cells from which they are derived, mainly utilize oxidative phosphorylation to produce ATP. Metabolic stress increases the rate of ATP production via oxidative phosphorylation and significantly reduces glycolysis in adult oligodendrocytes. The rate of ATP production was relatively unchanged in newly differentiated post natal oligodendrocytes under these stress conditions, while it was significantly reduced in oligodendrocyte progenitor cells. Our study indicates that both age and maturation influence the metabolic profile under optimal and stressed conditions, emphasizing the need to consider these variables for in vitro studies that aim to model adult human disease.

A novel function of TBK1 as a target of Cdon in oligodendrocyte differentiation and myelination.

During central nervous system development, oligodendrocyte progenitors elaborate multiple branched processes to contact axons and initiate myelination. Using cultured primary rat oligodendrocytes (OLGs), we have recently demonstrated that a cell surface protein belonging to the immunoglobulin superfamily, cell adhesion molecule-related, down-regulated by oncogenes (Cdon), is important in initiating OLG differentiation and axon myelination by promoting the formation of branched cellular processes; however, the molecular mechanism by which Cdon regulates OLG differentiation is not known. Here, using Cdon immunoprecipitation (IP) and liquid chromatography-tandem mass spectrometry analysis, we identified serine/threonine kinase TANK-binding kinase 1 (TBK1) as a candidate novel target of Cdon. We confirmed this interaction using co-IP and immunofluorescence with TBK1 antibodies, showing that TBK1 partly co-localizes with Cdon along cellular processes in puncta-like structures. We show that TBK1 is expressed throughout OLG differentiation, and surprisingly, that levels of phosphorylated TBK1 (ser172) increase during OLG maturation, while total levels of TBK1 protein decrease. To investigate function, TBK1 expression was knocked down using siRNA in OLG primary cultures, reducing protein levels by 69%. Two myelin-specific proteins, myelin basic protein and myelin-associated glycoprotein, were similarly reduced when examined at day 2 and day 4 of OLG differentiation. Reduced Cdon or TBK1 expression also decreased Akt phosphorylation at Threonine 308 in OLG. Our findings provide evidence that a Cdon-TBK1 complex is associated with Akt phosphorylation and early OLG differentiation.

Role of Sonic Hedgehog Signaling in Oligodendrocyte Differentiation.

During development, the secreted molecule Sonic Hedgehog (Shh) is required for lineage specification and proliferation of oligodendrocyte progenitors (OLPs), which are the glia cells responsible for the myelination of axons in the central nervous system (CNS). Shh signaling has been implicated in controlling both the generation of oligodendrocytes (OLGs) during embryonic development and their production in adulthood. Although, some evidence points to a role of Shh signaling in OLG development, its involvement in OLG differentiation remains to be fully determined. The objective of this study was to assess whether Shh signaling is involved in OLG differentiation after neural stem cell commitment to the OLG lineage. To address these questions, we manipulated Shh signaling using cyclopamine, a potent inhibitor of Shh signaling activator Smoothened (Smo), alone or combined with the agonist SAG in OLG primary cultures and assessed expression of myelin-specific markers. We found that inactivation of Shh signaling caused a dose-dependent decrease in myelin basic protein (MBP) and myelin associated glycoprotein (MAG) in differentiating OLGs. Co-treatment of the cells with SAG reversed the inhibitory effect of cyclopamine on both myelin-specific protein levels and morphological changes associated with it. Further experiments are required to elucidate the molecular mechanism by which Shh signaling regulates OLG differentiation.

Quaking Regulates Neurofascin 155 Expression for Myelin and Axoglial Junction Maintenance.

RNA binding proteins required for the maintenance of myelin and axoglial junctions are unknown. Herein, we report that deletion of the Quaking (QKI) RNA binding proteins in oligodendrocytes (OLs) using Olig2-Cre results in mice displaying rapid tremors at postnatal day 10, followed by death at postnatal week 3. Extensive CNS hypomyelination was observed as a result of OL differentiation defects during development. The QKI proteins were also required for adult myelin maintenance, because their ablation using PLP-CreERT resulted in hindlimb paralysis with immobility at ∼30 d after 4-hydroxytamoxifen injection. Moreover, deterioration of axoglial junctions of the spinal cord was observed and is consistent with a loss of Neurofascin 155 (Nfasc155) isoform that we confirmed as an alternative splice target of the QKI proteins. Our findings define roles for the QKI RNA binding proteins in myelin development and maintenance, as well as in the generation of Nfasc155 to maintain healthy axoglial junctions. SIGNIFICANCE STATEMENT: Neurofascin 155 is responsible for axoglial junction formation and maintenance. Using a genetic mouse model to delete Quaking (QKI) RNA-binding proteins in oligodendrocytes, we identify QKI as the long-sought regulator of Neurofascin alternative splicing, further establishing the role of QKI in oligodendrocyte development and myelination. We establish a new role for QKI in myelin and axoglial junction maintenance using an inducible genetic mouse model that deletes QKI in mature oligodendrocytes. Loss of QKI in adult oligodendrocytes leads to phenotypes reminiscent of the experimental autoimmune encephalomyelitis mouse model with complete hindlimb paralysis and death by 30 d after induction of QKI deletion.

Oligodendrogliopathy in Multiple Sclerosis: Low Glycolytic Metabolic Rate Promotes Oligodendrocyte Survival.

UNLABELLED: Multiple sclerosis (MS) lesions feature demyelination with limited remyelination. A distinct injury phenotype of MS lesions features dying back of oligodendrocyte (OL) terminal processes, a response that destabilizes myelin/axon interactions. This oligodendrogliopathy has been linked with local metabolic stress, similar to the penumbra of ischemic/hypoxic states. Here, we developed an in vitro oligodendrogliopathy model using human CNS-derived OLs and related this injury response to their distinct bioenergetic properties. We determined the energy utilization properties of adult human surgically derived OLs cultured under either optimal or metabolic stress conditions, deprivation of growth factors, and glucose and/or hypoxia using a Seahorse extracellular flux analyzer. Baseline studies were also performed on OL progenitor cells derived from the same tissue and postnatal rat-derived cells. Under basal conditions, adult human OLs were less metabolically active than their progenitors and both were less active than the rat cells. Human OLs and progenitors both used aerobic glycolysis for the majority of ATP production, a process that contributes to protein and lipid production necessary for myelin biosynthesis. Under stress conditions that induce significant process retraction with only marginal cell death, human OLs exhibited a significant reduction in overall energy utilization, particularly in glycolytic ATP production. The stress-induced reduction of glycolytic ATP production by the human OLs would exacerbate myelin process withdrawal while favoring cell survival, providing a potential basis for the oligodendrogliopathy observed in MS. The glycolytic pathway is a potential therapeutic target to promote myelin maintenance and enhance repair in MS. SIGNIFICANCE STATEMENT: The neurologic deficits that characterize multiple sclerosis (MS) reflect disruption of myelin (demyelination) within the CNS and failure of repair (remyelination). We define distinct energy utilization properties of human adult brain-derived oligodendrocytes and oligodendrocyte progenitor cells under conditions of metabolic stress that model the initial relapsing and subsequent progressive phases of MS. The observed changes in energy utilization affect both cell survival and myelination capacity. These processes may be amenable to therapeutic interventions to limit the extent of cumulative tissue injury and to promote repair in MS.

Cdon, a cell surface protein, mediates oligodendrocyte differentiation and myelination.

During central nervous system development, oligodendrocyte progenitors (OLPs) establish multiple branched processes and axonal contacts to initiate myelination. A complete understanding of the molecular signals implicated in cell surface interaction to initiate myelination/remyelination is currently lacking. The objective of our study was to assess whether Cdon, a cell surface protein that was shown to participate in muscle and neuron cell development, is involved in oligodendrocyte (OLG) differentiation and myelination. Here, we demonstrate that endogenous Cdon protein is expressed in OLPs, increasing in the early differentiation stages and decreasing in mature OLGs. Immunocytochemistry of endogenous Cdon showed localization on both OLG cell membranes and cellular processes exhibiting puncta- or varicosity-like structures. Cdon knockdown with siRNA decreased protein levels by 62% as well as two myelin-specific proteins, MBP and MAG. Conversely, overexpression of full-length rat Cdon increased myelin proteins in OLGs. The complexity of OLGs branching and contact point numbers with axons were also increased in Cdon overexpressing cells growing alone or in coculture with dorsal root ganglion neurons (DRGNs). Furthermore, myelination of DRGNs was decreased when OLPs were transfected with Cdon siRNA. Altogether, our results suggest that Cdon participates in OLG differentiation and myelination, most likely in the initial stages of development.

p38 Mitogen-Activated Protein Kinase Pathway Regulates Genes during Proliferation and Differentiation in Oligodendrocytes.

We have previously shown that p38 mitogen-activated protein kinase (p38 MAPK) is important for oligodendrocyte (OLG) differentiation and myelination. However, the precise cellular mechanisms by which p38 regulates OLG differentiation remain largely unknown. To determine whether p38 functions in part through transcriptional events in regulating OLG identity, we performed microarray analysis on differentiating oligodendrocyte progenitors (OLPs) treated with a p38 inhibitor. Consistent with a role in OLG differentiation, pharmacological inhibition of p38 down-regulated the transcription of genes that are involved in myelin biogenesis, transcriptional control and cell cycle. Proliferation assays showed that OLPs treated with the p38 inhibitor retained a proliferative capacity which could be induced upon application of mitogens demonstrating that after two days of p38-inhibition OLGs remained poised to continue mitosis. Together, our results suggest that the p38 pathway regulates gene transcription which can coordinate OLG differentiation. Our microarray dataset will provide a useful resource for future studies investigating the molecular mechanisms by which p38 regulates oligodendrocyte differentiation and myelination.

Purine-crosslinked injectable chitosan sponges promote oligodendrocyte progenitor cells' attachment and differentiation.

Oligodendrocyte Progenitor Cells (OPCs) reside in the central nervous system (CNS) and are responsible for remyelinating axons after a spinal cord injury (SCI). However, the remyelination process is incomplete and abnormal due to the inability of OPCs to fully differentiate at the site of injury. In this study a newly developed injectable chitosan sponge crosslinked using guanosine 5'-diphosphate (GDP) was used to enhance OPC survival, attachment and differentiation. This purine-based biomaterial is the first of its kind and its inception was based on the growing body of literature concerning the role of purinergic signalling in the CNS. GDP-crosslinked chitosan sponges are rapidly-gelling and can be easily administered in situ using an injection system based on a double-lumen design. The chitosan sponges prompted OPC differentiation even in the presence of mitogens. Moreover, neurotrophin-3 (NT-3) was successfully entrapped in the sponges and a sustained release for up to 30 days was achieved. OPCs were shown to differentiate into mature oligodendrocytes that express myelin basic protein (MBP) when cultured on sponges containing NT-3. These findings, along with the suitable physicochemical and biological properties, make these sponges conducive to use as viable therapeutic agents for enhancing remyelination post-SCI.

Properties of human central nervous system neurons in a glia-depleted (isolated) culture system.

BACKGROUND: Current methods for studying human neurons depend on a feeder layer of astroglia supplemented with animal serum to support the growing neurons. These requirements undermine many of the advantages provided by in vitro cell culture approaches when compared with more complex in vivo techniques. NEW METHOD: Here, we identified a reliable marker (MHCI) that allows for direct isolation of primary neurons from fetal human brain. We utilized a magnetic labeling and isolation technique to separate neurons from other neural cells. We established a defined condition, omitting the astroglial supports that could maintain the human neurons for varying amounts of time. RESULTS: We showed that the new method significantly improved the purity of human neurons in culture without the need for further chemical/mechanical enrichment. We demonstrated the suitability of these neurons for functional studies including Rho-kinase dependent regulation of neurite outgrowth and ensheathment in co-cultures with oligodendrocyte progenitor cells derived from fetal human brain. COMPARISON WITH EXISTING METHODS: The accountability for neuron-only seeding and the controllable density allows for better neuronal maturation and better visualization of the different neuronal compartments. The higher purity culture constitutes an effective system to study and screen for compounds that impact neuron biology without potential confounding effects from glial crowding. CONCLUSIONS: High purity human neurons generated using the improved method will enable enhanced reliability in the discovery and development of drugs with neuroregenerative and neuroprotective activity.

Role of p38MAPK in S1P receptor-mediated differentiation of human oligodendrocyte progenitors.

FTY720 is a sphingosine 1-phosphate receptor (S1PR) modulator used as a daily therapy to reduce disease activity in the relapsing form of multiple sclerosis (MS). FTY720 readily accesses the CNS. Previous studies have shown that phosphorylated FTY720 (FTY720-p) enhances oligodendrocyte progenitor cell (OPC) survival, differentiation, and remyelination following experimentally induced demyelination in rodents. To elucidate the underlying mechanism, human fetal OPCs alone or in co-culture with rat dorsal root ganglia neurons (DRGN) were treated daily with FTY720-p, a condition that desensitizes cellular responses to S1P, the natural ligand of S1PR. In co-cultures, FTY720-p and S1P given daily or every three days increased the number of O1/MBP double positive cells and axonal ensheathment. In cultures composed of PDGFRα-antibody selected cells alone, daily application of FTY720-p also increased the number of O4/GC double positive cells. At an early time point (day 2), FTY720-p activated ERK1/2, CREB and p38MAPK in O4-positive cells, as well as in ß-III Tubulin positive neurons and GFAP positive astrocytes. In later cultures (day 6), FTY720-p activated p38MAPK in O4 positive cells, p38MAPK and ERK1/2 in neurons, and p38MAPK, ERK1/2 and CREB in astrocytes. A MEK inhibitor (U0126) prevented the differentiation of OPCs into O4-positive cells, while a p38MAPK inhibitor (PD169316) blocked progression into O4-positive and into GC-positive stages of differentiation. Our results demonstrate that FTY720-p, under conditions that model daily clinical use, can act directly on OPCs to impact differentiation, and also indirectly via neurons and astrocytes by activating ERK1/2 and p38MAPK.

Agonist-induced down-regulation of AMPA receptors in oligodendrocyte progenitors.

Prolonged exposure of oligodendrocyte progenitor cultures to non-toxic concentrations of glutamate receptor agonists for 24 h decreased cellular proliferation mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Since prolonged agonist stimulation can regulate the expression of various families of receptors, we examined this possibility. Pretreatment of progenitor cultures with 100 µM kainic acid (KA) for 1-24 h caused a time-dependent decrease in AMPA receptor activity, determined by agonist-induced (45)Ca(2+) uptake. The maximum effect (70-80% decrease), observed in the 24 h-pretreated cells, was accompanied by a significant reduction in AMPA receptor subunits, as determined by Western blotting. GluR2/3 and GluR4 subunits were the most affected. Receptor down-regulation and (45)Ca(2+) uptake were only partially reversible upon KA removal. Furthermore, 24 h co-treatment of cultures with CNQX blocked the KA-induced decreases in calcium uptake. To address whether calpain, a calcium-activated protease, was implicated in the regulation of the AMPA receptor subunits, cultures were treated with the specific inhibitor PD150606 alone or in combination with KA for 24 h. Calpain inhibition significantly increased GluR1 in both conditions and partly reversed downregulation of GluR4 by KA. Collectively, these results indicate that calpain is not involved in the agonist-induced down-regulation of AMPA receptors subunits 2/3 in oligodendrocyte progenitors, while it downregulates GluR1 and GluR4.

The PTEN inhibitor bisperoxovanadium enhances myelination by amplifying IGF-1 signaling in rat and human oligodendrocyte progenitors.

Oligodendrocytes (OLGs) produce and maintain myelin in the central nervous system (CNS). In the demyelinating autoimmune disease multiple sclerosis, OLGs are damaged and those remaining fail to fully remyelinate CNS lesions. Therefore, current therapies directed to restrain the inflammation process with approaches that protect and reconstitute oligodendrocyte density would be essential to pave the way of myelin repair. A critical signal for oligodendrocytes is insulin-like growth factor-1 (IGF-1), which promotes their development and ultimately myelin formation. PTEN inhibits the phosphoinositide 3-kinase (PI3K)/Akt signaling, a convergence downstream pathway for growth factors such as IGF-1. In this report, we temporarily inhibited PTEN activity by treating rat and human oligodendrocyte progenitors (OLPs) cultured alone or with dorsal root ganglion neurons (DRGNs) with bisperoxovanadium (phen). Our findings show that phen potentiates IGF-1 actions by increasing proliferation of OLPs in a concentration-dependent manner, and caused a sustained and time-dependent activation of the main pathways: PI3K/Akt/mammalian target of rapamycin (mTOR) and MEK/ERK. At low concentrations, IGF-1 and phen stimulated the differentiation of rat and human OLPs. Concordantly, the PTEN inhibitor together with IGF-1 robustly augmented myelin basic protein accumulation in rat newborn and human fetal OLGs co-cultured with DRGNs in a longer timeframe by promoting the elaboration of organized myelinated fibers as evidenced by confocal microscopy. Thus, our results suggest that a transient suppression of a potential barrier for myelination in combination with other therapeutic approaches including growth factors may be promising to improve the functional recovery of CNS injuries.

A miniaturized multipurpose platform for rapid, label-free, and simultaneous separation, patterning, and in vitro culture of primary and rare cells.

Given that current cell isolation techniques are expensive, time consuming, yield low isolation purities, and/or alter target cell properties, a versatile, cost effective, and easy-to-operate microchip with the capability to simultaneously separate, capture, pattern, and culture rare and primary cells in vitro is developed. The platform is based on target cell adhesion onto the micro-fabricated interfaces produced by microcontact printing of cell-specific antibodies. Results show over 95% separation efficiency in less than 10 min for the separation of oligodendrocyte progenitor cells (OPCs) and cardiomyocytes from rat brain and heart mixtures, respectively. Target cell attachment and single cell spreading can be precisely controlled on the basis of the designed patterns. Both cell types can maintain their biofunctionality. Indeed, isolated OPCs can proliferate and differentiate into mature oligodendrocytes, while isolated cardiomyocytes retain their contractile properties on the separation platform. Successful separation of two dissimilar cell types present in varying concentrations in their respective cell mixtures and the demonstration of their integrity after separation open new avenues for time and cost-effective sorting of various cell types using the developed miniaturized platform.

Oligodendrocyte progenitor cell susceptibility to injury in multiple sclerosis.

Remyelination in multiple sclerosis (MS) is often incomplete. In experimental models, oligodendrocyte progenitor cells (OPCs) rather than previously myelinating oligodendrocytes (OLs) are responsible for remyelination. This study compares the relative susceptibility of adult human OPCs and mature OLs to injury in actively demyelinating MS lesions and under in vitro stress conditions. In all lesions (n = 20), the number of OLs (Olig2 weak/NogoA positive) was reduced compared to control white matter (mean 38 ± 4% of control value). In 11 cases, OPC numbers (Olig2 strong; NogoA negative) were also decreased; in eight of these, the reduction was greater for OPCs than for OLs. In the other nine samples, OPC numbers were greater than control white matter, indicating ongoing OPC migration and/or proliferation. Analysis of co-cultures with rat dorsal root ganglia neurons confirmed that OPCs were more capable of contacting and ensheathing axons than OLs. In isolated culture under stress conditions (withdrawal of serum/glucose and/or antioxidants), OPCs showed increased cell death and reduced process extension compared to OLs. Under all culture conditions, OPCs up-regulated expression of genes in the extrinsic proapoptotic pathway, and had increased susceptibility to tumor necrosis factor-induced cell death as compared to OLs. Our data suggest that susceptibility of OPCs to injury within the MS lesion environment contributes to the limited remyelination in MS.

Rapid, guanosine 5'-diphosphate-induced, gelation of chitosan sponges as novel injectable scaffolds for soft tissue engineering and drug delivery applications.

Novel injectable chitosan sponges based on rapid ionic crosslinking using guanosine 5'-diphosphate are introduced. The rapid gelation, high water retention, desirable physicochemical properties, soft tissue-like mechanical properties, and excellent cytocompatibility make these injectable sponges promising candidates for tissue regeneration and drug delivery applications.

Mitogen-activated protein kinase p38 regulates Krox-20 to direct Schwann cell differentiation and peripheral myelination.

We previously reported that addition of extracellular matrix (ECM) extracts to rat Schwann cell-dorsal root ganglion neuron (DRGN) co-cultures activated mitogen-activated protein kinase (MAPK) p38, whereas inhibition blocked myelination. Here, we used p38 pharmacological inhibitors and gene silencing to assess their effects on downstream kinases and key transcription factors. We show that p38α regulates expression of the master transcription factor, Krox-20, required for the onset of myelination in Schwann cell-DRGNs, as assessed by immunocytochemistry and qRT-PCR. p38 activity is also required for the expression of the cell cycle inhibitor p27(kip1) , associated with Schwann cell differentiation. Three potential effectors of p38 were explored: MAPK-activated protein kinase-2 (MK2), mitogen and stress-activated protein kinase-1 (MSK-1), and the transcription factor cAMP response element-binding protein (CREB). Inhibition of MK2 with CMPD1 or gene knockdown with siRNAs reduced numbers of Krox-20-positive Schwann cells and expression of myelin proteins MBP and MAG. ECM activated CREB and increased Krox-20 expression, whereas CREB1 gene silencing reduced Krox-20. Furthermore, two nonselective inhibitors of MSK-1 (H89 and R0-318820) decreased ECM-induced CREB phosphorylation and, similar to anti-MSK-1 siRNAs, reduced Krox-20-positive cells. In addition, p38 modulated the expression of two transcription factors involved in the regulation of Krox-20 [suppressed cAMP-inducible protein (SCIP) and Sox10], but not Sox2, an antagonist of Krox-20. Collectively, our results show that p38 primarily directs Schwann cell differentiation and peripheral myelination by regulating Krox-20 expression through its downstream effectors, MK2 and MSK-1/CREB, and transcription factors SCIP and Sox10.

Oligodendrocyte-protection and remyelination post-spinal cord injuries: a review.

In the past four decades, the main focus of investigators in the field of spinal cord regeneration has been to devise therapeutic measures that enhance neural regeneration. More recently, emphasis has been placed on enhancing remyelination and providing oligodendrocyte-protection after a spinal cord injury (SCI). Demyelination post-SCI is part of the cascading secondary injury that takes place immediately after the primary insult; therefore, therapeutic measures are needed to reduce oligodendrocyte death and/or enhance remyelination during the acute stage, preserving neurological functions that would be lost otherwise. In this review a thorough investigation of the oligodendrocyte-protective and remyelinative molecular therapies available to date is provided. The advent of new biomaterials shown to promote remyelination post-SCI is discussed mainly in the context of a combinatorial approach where the biomaterial also provides drug delivery capabilities. The aim of these molecular and biomaterial-based therapies is twofold: (1) oligodendrocyte-protective therapy, which involves protecting already existing oligodendrocytes from undergoing apoptosis/necrosis; and (2) inductive remyelination, which involves harnessing the remyelinative capabilities of endogenous oligodendrocyte precursor cells (OPCs) at the lesion site by providing a suitable environment for their migration, survival, proliferation and differentiation. From the evidence reported in the literature, we conclude that the use of a combinatorial approach including biomaterials and molecular therapies would provide advantages such as: (1) sustained release of the therapeutic molecule, (2) local delivery at the lesion site, and (3) an environment at the site of injury that promotes OPC migration, differentiation and remyelination.

Human fetal oligodendrocyte progenitor cells from different gestational stages exhibit substantially different potential to myelinate.

To investigate age-related intrinsic regulation of the capacity of human fetal oligodendrocyte progenitor cells (OPCs) to myelinate, potential OPCs were selected from 15- to 23-gestational-week (gw) human fetal brain tissue based on the expression of gangliosides--recognized with the monoclonal antibody A2B5, which detects multipotent cells including OPCs--or platelet-derived growth factor receptor α (PDGFRα), an early marker of the oligodendroglial lineage. Cells were either cultured alone or cocultured with rat dorsal root ganglia neurons (DRGNs). When cultured alone, both the A2B5- and PDGFRα-selected cells exhibited age-dependent increases in early to mid-stage lineage markers, including sulfatides (O4 antibody) and the transcription factor Olig2, while the cell death rate correlated negatively with age. In coculture with neurons, cells also expressed the myelin components galactocerebroside (GC) and myelin basic protein (MBP), and ensheathed axons. In DRGN cocultures, A2B5+ cells derived from >19 gw produced more GC+/MBP+ cells compared with the 15-17-week cells. The number of GC+ cells making axonal contacts, and ensheathing axonal segments per cell increased proportionally to gestational age. This age-dependent difference in GC/MBP cell number and capacity to ensheath axons persisted when PDGFRα selection was used to enrich for the number of OPCs in cultures derived from younger ages. Addition of the growth factors brain-derived neurotrophic factor (BDNF) and insulin-like growth factor 1 (IGF-1) enhanced OPC differentiation under all conditions. These findings indicate that intrinsic regulatory mechanisms associated with the chronological age of the donor cells are key variables to assess when considering the myelination capacity of OPCs for cellular replacement therapy.

Type II arginine methyltransferase PRMT5 regulates gene expression of inhibitors of differentiation/DNA binding Id2 and Id4 during glial cell differentiation.

PRMT5 is a type II protein arginine methyltranferase that catalyzes monomethylation and symmetric dimethylation of arginine residues. PRMT5 is functionally involved in a variety of biological processes including embryo development and circadian clock regulation. However, the role of PRMT5 in oligodendrocyte differentiation and central nervous system myelination is unknown. Here we show that PRMT5 expression gradually increases throughout postnatal brain development, coinciding with the period of active myelination. PRMT5 expression was observed in neurons, astrocytes, and oligodendrocytes. siRNA-mediated depletion of PRMT5 in mouse primary oligodendrocyte progenitor cells abrogated oligodendrocyte differentiation. In addition, the PRMT5-depleted oligodendrocyte progenitor and C6 glioma cells expressed high levels of the inhibitors of differentiation/DNA binding, Id2 and Id4, known repressors of glial cell differentiation. We observed that CpG-rich islands within the Id2 and Id4 genes were bound by PRMT5 and were hypomethylated in PRMT5-deficient cells, suggesting that PRMT5 plays a role in gene silencing during glial cell differentiation. Our findings define a role of PRMT5 in glial cell differentiation and link PRMT5 to epigenetic changes during oligodendrocyte differentiation.