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.

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.

Objective: To determine the relationship between the bio-energetic properties of oligodendocytes (OLs) and their response to metabolic stresses that mimic the multiple sclerosis (MS) lesion micro-environment. Background: The production of myelin by OLs is a highly energetic process, requiring mitochondrial oxidative phosphorylation (OXPHOS) for ATP production and glycolysis/lactate to provide the carbon backbones necessary for lipid biosynthesis. Oligodendrogliopathy, which features the dying back of oligodendrocyte terminal processes, is a distinct type of injury response observed in MS lesions. This dying back process in MS has been linked to metabolic stress, which in MS lesions reflect disturbed micro-circulation with associated ischemia and/or production of toxic metabolites that disturb mitochondrial energy metabolism (Lassmann 2003). Methods: We used OLs isolated from adult human brain specimens to examine their injury responses (survival, process extension) to reduced trophic factor, low glucose, and hypoxia conditions as a model of oligodendrogliopathy observed in MS lesions. We further examined the underlying bio-energetic properties of OLs using a Seahorse XF96 extracellular flux analyzer under the basal, metabolic stress and recovery conditions. Results: Human OLs grown under the above stress conditions for 48 hours showed only a marginal increase in [percnt] of TUNEL+ cells but a significant decrease in process area per cell. Basal aerobic glycolysis under optimal conditions provides the majority of ATP production in OLs. Under stress conditions, this ATP production is significantly decreased. Removal of stress conditions for 48 hours allowed for recovery of ATP production that occurred under the metabolic stress conditions. Conclusions: Oligodendrocytes under metabolic stress conditions show significant reduction in glycolytic ATP production that is required for myelin maintenance. Here we show that the restoration of optimal metabolic conditions can restore energy production in human OLs. It remains to be determined when such intervention will result in long-term cell survival. Disclosure: Dr. Cui has nothing to disclose. Dr. Rone has nothing to disclose. Dr. Khan has nothing to disclose. Dr. Bedard has nothing to disclose. Dr. Almazan has nothing to disclose. Dr. Ludwin has nothing to disclose. Dr. Kennedy has nothing to disclose. Dr. Antel has received compensation for activities with Novartis, Sanofi/Genzyme Inc., Biogen Idec, EMD Serono, Juno Therapeutics, and MedDay for serving on the advisory boards. Dr. Antel has received personal compensation in an editorial capacity for Mul

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) usingOlig2–Creresults 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 usingPLP–CreERTresulted 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 STATEMENTNeurofascin 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.

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.

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.

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.

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. (C) 2015 Elsevier B.V. All rights reserved.

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.

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.

With the death of Theodore L. Sourkes on Jan. 17, 2015, at age 95, the CCNP, Canada and the international research community lost one of the pioneers of a research discipline: the study of the biochemistry of the brain in relation to mental and neurological disorders. Ted was born in Montréal, Que

Prolonged exposure of oligodendrocyte progenitor cultures to non-toxic concentrations of glutamate receptor agonists for 24 h decreased cellular proliferation mediated by alpha-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 mu M kainic acid (KA) for 1-24 h caused a time-dependent decrease in AMPA receptor activity, determined by agonist-induced Ca-45(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 Ca-45(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. (C) 2014 Elsevier Ltd. All rights reserved.

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 β-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.

We developed a rapidly-gelling chitosan sponge crosslinked with Guanosine 5'-Diphosphate (GDP). GDP has not been previously explored as an anionic crosslinker, and it was used in this application since the nucleoside guanosine has been shown to improve remyelination in situ , and thus its presence in the sponge composition was hypothesized to induce Oligodendrocyte Progenitor Cells' (OPC) differentiation. In addition to the chemical composition tailored to target OPCs, the developed chitosan sponge possesses a wide range of desirable physicochemical properties such as: rapid gelation, high porosity with interconnected pores, moduli of elasticity resembling that of soft tissue and cytocompatibility with many cell types. Moreover, protein encapsulation into the sponges was possible with high encapsulation efficiencies (e.g. BMP-7 and NT-3). In this study, BDNF was encapsulated in the chitosan sponges with an encapsulation efficiency greater than 80% and a sustained release over a 16-day period was achieved. We demonstrate here for the first time, the attachment of human fetal OPCs to the sponges and their differentiation after 12 days of culture. Overall, this newly-introduced injectable sponge is a promising therapeutic modality that can be used to enhance remyelination post-spinal cord injuries.

OBJECTIVE:To elucidate the molecular signaling mechanisms mediating FTY720p effects on human fetal oligodendrocyte progenitor cells (OPCs) differentiation and axonal ensheathment. BACKGROUND:FTY720 (Fingolimod) is a clinically approved sphingosine-1-phosphate receptor (S1PR) modulator which readily enters the CNS. FTY720 phosphate (FTY720p), the active moiety of FTY720 which undergoes phosphorylation in vivo, modulates initial process outgrowth in human progenitor and mature oligodendrocyte cultures and enhances differentiation and axonal ensheathment of OPCs when co-cultured with rat dorsal root ganglia neurons (DRGNs). Although repeated daily application of FTY720p inhibits cell signaling and/or functional responses mediated by the natural ligand S1P in human astrocytes and oligodendrocytes, FTY720p can continue to induce persistent receptor-mediated activation of signaling pathways. DESIGN/METHODS:OPCs were selected from 15-18 gestational week human brain samples using PDGFRα- or A2B5- antibody-coated immuno-magnetic beads. FTY720p or S1P (both at 50 nM) were added either daily or every 2 days. Phosphorylation of ERK1/2, p38MAPK or CREB was determined by Western blotting or immuno-staining together with cell type-specific markers. RESULTS:At an early time point (day 2), FTY720-p activated ERK1/2, p38MAPK and CREB in O4 positive cells, as well as in beta-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. O4 cells in later cultures (day 6) are morphologically more differentiated compared to early time point cultures. 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 galactocerebroside (GC) positive stages of differentiation. CONCLUSIONS:Our results demonstrate that FTY720p, under conditions that model clinical use, can act directly on OPCs to impact differentiation, and also indirectly via neurons and astrocytes by activating ERK1/2 and p38MAPK. Study Supported by:MS Society of Canada; CIHR (Industry partnership program); Novartis.