Oligodendrocyte Process Extension Research Articles

Ephrin-A1-EphA4 signaling negatively regulates myelination in the central nervous system.

During development of the central nervous system not all axons are myelinated, and axons may have distinct myelination patterns. Furthermore, the number of myelin sheaths formed by each oligodendrocyte is highly variable. However, our current knowledge about the axo-glia communication that regulates the formation of myelin sheaths spatially and temporally is limited. By using axon-mimicking microfibers and a zebrafish model system, we show that axonal ephrin-A1 inhibits myelination. Ephrin-A1 interacts with EphA4 to activate the ephexin1-RhoA-Rock-myosin 2 signaling cascade and causes inhibition of oligodendrocyte process extension. Both in myelinating co-cultures and in zebrafish larvae, activation of EphA4 decreases myelination, whereas myelination is increased by inhibition of EphA4 signaling at different levels of the pathway, or by receptor knockdown. Mechanistically, the enhanced myelination is a result of a higher number of myelin sheaths formed by each oligodendrocyte, not an increased number of mature cells. Thus, we have identified EphA4 and ephrin-A1 as novel negative regulators of myelination. Our data suggest that activation of an EphA4-RhoA pathway in oligodendrocytes by axonal ephrin-A1 inhibits stable axo-glia interaction required for generating a myelin sheath.

Integrin-Linked Kinase (ILK) Deletion Disrupts Oligodendrocyte Development by Altering Cell Cycle

During development, oligodendrocytes are initially specified, after which oligodendrocyte precursor cells (OPCs) migrate and proliferate before differentiating into myelinating cells. Lineage-specific programming of oligodendrocytes results from sensing environmental cues through membrane-bound receptors and related intracellular signaling molecules. Integrin-linked kinase (ILK) is an important protein that is expressed at the inner margins of the plasma membrane and can mediate some of these signals. The current studies demonstrate that ILK deletion reduces the proliferation and differentiation of OPCs in the developing CNS. There was a significant decrease in the number of OPCs and mature oligodendrocytes throughout postnatal development in Olig1Cre^+/− × ILK^fl/fl mice. These changes were accompanied by reduced numbers of myelinated axons. Key proteins involved in cell cycle regulation were dysregulated. Cyclin D1/D3 and cyclin-dependent kinase 2/4 (cdc2/cdc4) were downregulated and the cell cycle inhibitor protein p27 Kip1 was upregulated. Therefore, ILK deletion impaired the developmental profile, proliferation, and differentiation of OPCs by altering the expression of regulatory cytoplasmic and nuclear factors. <b>SIGNIFICANCE STATEMENT</b> Integrin-linked kinase (ILK) is a scaffolding protein involved in integrating signals from the extracellular environment and communicating those signals to downstream effectors within cells. It has been proposed to regulate aspects of oligodendrocyte process extension and thereby myelination. However, the current studies demonstrate that it has an earlier impact on cells in this lineage. Knocking down ILK in Olig1-Cre-expressing cells reduces the pool of oligodendrocyte progenitor cells (OPCs). This smaller pool of OPCs results from altered cell cycle and reduced cell proliferation. These cells myelinate fewer axons than in wild-type mice and, in corpus callosum, the myelin is thinner than in controls. Interestingly, the smaller pool of spinal cord oligodendrocytes generates myelin that is of normal thickness.

Integrin-Linked Kinase (ILK) Deletion Disrupts Oligodendrocyte Development by Altering Cell Cycle

Integrin-linked kinase (ILK) is a scaffolding protein involved in integrating signals from the extracellular environment and communicating those signals to downstream effectors within cells. It has been proposed to regulate aspects of oligodendrocyte process extension and thereby myelination. However, the current studies demonstrate that it has an earlier impact on cells in this lineage. Knocking down ILK in Olig1-Cre-expressing cells reduces the pool of oligodendrocyte progenitor cells (OPCs). This smaller pool of OPCs results from altered cell cycle and reduced cell proliferation. These cells myelinate fewer axons than in wild-type mice and, in corpus callosum, the myelin is thinner than in controls. Interestingly, the smaller pool of spinal cord oligodendrocytes generates myelin that is of normal thickness.

Axo-Glia Interaction Preceding CNS Myelination Is Regulated by Bidirectional Eph-Ephrin Signaling

In the central nervous system, myelination of axons is required to ensure fast saltatory conduction and for survival of neurons. However, not all axons are myelinated, and the molecular mechanisms involved in guiding the oligodendrocyte processes toward the axons to be myelinated are not well understood. Only a few negative or positive guidance clues that are involved in regulating axo-glia interaction prior to myelination have been identified. One example is laminin, known to be required for early axo-glia interaction, which functions through α6β1 integrin. Here, we identify the Eph-ephrin family of guidance receptors as novel regulators of the initial axo-glia interaction, preceding myelination. We demonstrate that so-called forward and reverse signaling, mediated by members of both Eph and ephrin subfamilies, has distinct and opposing effects on processes extension and myelin sheet formation. EphA forward signaling inhibits oligodendrocyte process extension and myelin sheet formation, and blocking of bidirectional signaling through this receptor enhances myelination. Similarly, EphB forward signaling also reduces myelin membrane formation, but in contrast to EphA forward signaling, this occurs in an integrin-dependent manner, which can be reversed by overexpression of a constitutive active β1-integrin. Furthermore, ephrin-B reverse signaling induced by EphA4 or EphB1 enhances myelin sheet formation. Combined, this suggests that the Eph-ephrin receptors are important mediators of bidirectional signaling between axons and oligodendrocytes. It further implies that balancing Eph-ephrin forward and reverse signaling is important in the selection process of axons to be myelinated.

Inhibitors of myelination: ECM changes, CSPGs and PTPs

After inflammation-induced demyelination, such as in the disease multiple sclerosis, endogenous remyelination often fails. However, in animal models of demyelination induced with toxins, remyelination can be quite robust. A significant difference between inflammation-induced and toxin-induced demyelination is the response of local cells within the lesion, including astrocytes, oligodendrocytes, microglia/macrophages, and NG2+ cells, which respond to inflammatory stimuli with increased extracellular matrix (ECM) protein and chondroitin sulfate proteoglycan (CSPG) production and deposition. Here, we summarize current knowledge of ECM changes in demyelinating lesions, as well as oligodendrocyte responses to aberrant ECM proteins and CSPGs after various types of demyelinating insults. The discovery that CSPGs act through the receptor protein tyrosine phosphatase sigma (PTPσ) and the Rho-ROCK pathway to inhibit oligodendrocyte process extension and myelination, but not oligodendrocyte differentiation (Pendleton et al., Experimental Neurology (2013) vol. 247, pp. 113–121), highlights the need to better understand the ECM changes that accompany demyelination and their influence on oligodendrocytes and effective remyelination.

A central role for RhoA during oligodendroglial maturation in the switch from Netrin‐1‐mediated chemorepulsion to process elaboration

The guidance cue netrin-1 and its receptor Deleted in Colorectal Cancer play distinct roles during different stages of oligodendrocyte development. A gradient of netrin-1 repels migrating oligodendrocyte precursor cells (OPCs) in the embryonic spinal cord by promoting process collapse, but later in development netrin-1 increases oligodendrocyte process extension and branching. Here we investigate the intracellular mechanism that governs this switch in response to netrin-1, and focus on the role of the GTPase RhoA and its effector Rho Kinase (ROCK) downstream of netrin-1 in OPCs and maturing oligodendrocytes. In OPCs, we show that netrin-1 induces a sustained increase in RhoA activity that requires Deleted in Colorectal Cancer function. Furthermore, we demonstrate that activation of RhoA and ROCK is required for the reduction in OPC process length triggered by netrin-1, and for the chemorepellent response made by OPCs to netrin-1. Unlike OPCs, application of netrin-1 to oligodendrocytes decreases RhoA activity. We demonstrate that inactivation of RhoA is essential for netrin-1 to increase oligodendrocyte process branching. We conclude that netrin-1 induces distinct morphological responses in OPCs and oligodendrocytes through differential regulation of RhoA activity.

Subcellular localization of Mayven following expression of wild type and mutant EGFP tagged cDNAs

BackgroundProcess formation by glial cells is crucial to their function. Mayven, an actin binding, multi-domain polypeptide, and member of the BTB-BACK-Kelch family have been shown to be important in oligodendrocyte process extension. To assess the role of Mayven in neural cell process extension we have tracked the subcellular distribution of exogenous Mayven following expression of a rat Mayven -EGFP cDNA in a variety of neural cell backgrounds and specifically in OEC tranfectants following drug treatment to disrupt the integrity of the cytoskeleton. A comparison was made between the subcellular localization following transient transfection of OECs with full-length Mayven cDNA and a series of mutant domain constructs.ResultsThe subcellular location of Mayven in OEC transfectants showed a characteristic distribution with intense foci of staining towards the process tips corresponding to regions of accumulated Mayven overlapping in part with lammelipodial actin and was absent from the filipodia and the outer membrane. This signature pattern was also observed in Schwann cells, Oli-Neu cells, astrocytes and the neuroblastoma cell line B104 transfectants and resembled the exogenous and endogenous Mayven distribution in oligodendrocytes. This contrasted with the localization pattern in non-neural cells. There was a re-localization of Mayven in OEC transfectants following drug treatment to challenge the integrity of the actin cytoskeleton while breakdown of the microtubular component had no discernible impact on the accumulation of Mayven in the process tips. Deletion of the first three amino acids of the SH3 motif of the putative Fyn Kinase binding domain at the amino terminus significantly compromised this signature pattern as did the removal of the last Kelch repeat unit of six unit Kelch domain comprising the carboxyl terminus. In addition, there was a reduction in process length in mutant transfectants. Co-expression studies with a haemagglutinin (HA) tagged wild type Mayven cDNA and EGFP tagged mutant cDNAs suggested a homomeric interaction mediated by the BTB/POZ domain.ConclusionsExogenous Mayven is transported to the lamellipodia in neural transfectants associating with the actin cytoskeletal network. In addition to the importance of the internal BTB/POZ domain, this subcellular distribution pattern is dependent on the presence of an intact amino and carboxyl terminus.

Astrocytes, but not olfactory ensheathing cells or Schwann cells, promote myelination of CNS axons in vitro

We have examined the interaction between olfactory ensheathing cells (OECs), Schwann cells (SC), oligodendrocytes, and CNS axons using cultures generated from embryonic rat spinal cord. Oligodendrocyte process extension and myelination in these cultures was poor if the cells were plated on OECs or SCs. Myelin internodes and nodes of Ranvier formed frequently if these cultures were plated onto monolayers of neurosphere-derived astrocytes (NsAs). In the myelinated fibers generated on NsAs, Nav channels, caspr, and neurofascin molecules were correctly assembled at the nodes of Ranvier. The density of neurites, survival, and antigenic differentiation of oligodendrocytes was similar on OEC and NsAs monolayers. However, on OEC monolayers, despite a transient increase in the number of endogenous oligodendrocytes, there was a decrease in oligodendrocyte process extension and axonal ensheathment when compared with cultures plated on NsAs monolayers. To determine if these changes were due to axonal or glial factors, spinal cord oligodendrocytes were plated onto monolayers of OECs, NsAs, and poly-L-lysine in the absence of neurons. In these cultures, process extension and myelin-like membrane formation by oligodendrocytes was improved on monolayers of OEC. This suggests that inhibition of process extension is mediated via cross-talk between OECs and neurites. In cultures containing axons plated on OEC monolayers, oligodendrocyte process formation, axonal ensheathment, and myelination occurred albeit lower if the cultures were supplemented with NsAs conditioned medium. These data suggest OECs can permit neurite extension and oligodendrocyte proliferation, but lack secreted factor(s) and possible cell-cell contact that is necessary for oligodendrocyte process extension and myelination.

Isolation and Expression Pattern of Human Unc-33-Like Phosphoprotein 6/Collapsin Response Mediator Protein 5 (Ulip6/CRMP5): Coexistence with Ulip2/CRMP2 in Sema3A- Sensitive Oligodendrocytes

The Unc-33-like phosphoprotein/collapsin response mediator protein (Ulip/CRMP) family consists of four homologous phosphoproteins considered crucial for brain development. Autoantibodies produced against member(s) of this family by patients with paraneoplastic neurological diseases have made it possible to clone a fifth human Ulip/CRMP and characterize its cellular and anatomical distribution in developing brain. This protein, referred to as Ulip6/CRMP5, is highly expressed during rat brain development in postmitotic neural precursors and in the fasciculi of fibers, suggesting its involvement in neuronal migration/differentiation and axonal growth. In the adult, Ulip6/CRMP5 is still expressed in some neurons, namely in areas that retain neurogenesis and in oligodendrocytes in the midbrain, hindbrain, and spinal cord. Ulip2/CRMP2 and Ulip6/CRMP5 are coexpressed in postmitotic neural precursors at certain times during development and in oligodendrocytes in the adult. Because Ulip2/CRMP2 has been reported to mediate semaphorin-3A (Sema3A) signal in developing neurons, in studies to understand the function of Ulip6/CRMP5 and Ulip2/CRMP2 in the adult, purified adult rat brain oligodendrocytes were cultured in a Sema3A-conditioned medium. Oligodendrocytes were found to have Sema3A binding sites and to express neuropilin-1, the major Sema3A receptor component. In the presence of Sema3A, these oligodendrocytes displayed a dramatic reduction in process extension, which was reversed by removal of Sema3A and prevented by anti-neuropilin-1, anti-Ulip6/CRMP5, anti-Ulip2/CRMP2 antibodies, or VEGF-165, another neuropilin-1 ligand. These results indicate the existence in the adult brain of a Sema3A signaling pathway that modulates oligodendrocyte process extension mediated by neuropilin-1, Ulip6/CRMP5, and Ulip2/CRMP2, and they open new fields of investigation of neuron/oligodendrocyte interactions in the normal and pathological brain.