Lysophosphatidic acid signaling via LPA6 : A negative modulator of developmental oligodendrocyte maturation.

The developmental process of central nervous system (CNS) myelin sheath formation is characterized by well-coordinated cellular activities ultimately ensuring rapid and synchronized neural communication. During this process, myelinating CNS cells, namely oligodendrocytes (OLGs), undergo distinct steps of differentiation, whereby the progression of earlier maturation stages of OLGs represents a critical step toward the timely establishment of myelinated axonal circuits. Given the complexity of functional integration, it is not surprising that OLG maturation is controlled by a yet fully to be defined set of both negative and positive modulators. In this context, we provide here first evidence for a role of lysophosphatidic acid (LPA) signaling via the G protein-coupled receptor LPA6 as a negative modulatory regulator of myelination-associated gene expression in OLGs. More specifically, the cell surface accessibility of LPA6 was found to be restricted to the earlier maturation stages of differentiating OLGs, and OLG maturation was found to occur precociously in Lpar6 knockout mice. To further substantiate these findings, a novel small molecule ligand with selectivity for preferentially LPA6 and LPA6 agonist characteristics was functionally characterized in vitro in primary cultures of rat OLGs and in vivo in the developing zebrafish. Utilizing this approach, a negative modulatory role of LPA6 signaling in OLG maturation could be corroborated. During development, such a functional role of LPA6 signaling likely serves to ensure timely coordination of circuit formation and myelination. Under pathological conditions as seen in the major human demyelinating disease multiple sclerosis (MS), however, persistent LPA6 expression and signaling in OLGs can be seen as an inhibitor of myelin repair. Thus, it is of interest that LPA6 protein levels appear elevated in MS brain samples, thereby suggesting that LPA6 signaling may represent a potential new druggable pathway suitable to promote myelin repair in MS.

Astrocytes as a target for Nogo-A and implications for synapse formation in vitro and in a model of acute demyelination.

Central nervous system (CNS) function depends on precise synaptogenesis, which is shaped by environmental cues and cellular interactions. Astrocytes are outstanding regulators of synapse development and plasticity through contact-dependent signals and through the release of pro- and antisynaptogenic factors. Conversely, myelin and its associated proteins, including Nogo-A, affect synapses in a inhibitory fashion and contribute to neural circuitry stabilization. However, the roles of Nogo-A-astrocyte interactions and their implications in synapse development and plasticity have not been characterized. Therefore, we aimed to investigate whether Nogo-A affects the capacity of astrocytes to induce synaptogenesis. Additionally, we assessed whether downregulation of Nogo-A signaling in an in vivo demyelination model impacts the synaptogenic potential of astrocytes. Our in vitro data show that cortical astrocytes respond to Nogo-A through RhoA pathway activation, exhibiting stress fiber formation and decreased ramified morphology. This phenotype was associated with reduced levels of GLAST protein and aspartate uptake, decreased mRNA levels of the synaptogenesis-associated genes Hevin, glypican-4, TGF-ß1 and BDNF, and decreased and increased protein levels of Hevin and SPARC, respectively. Corroborating these findings, conditioned medium from Nogo-A-treated astrocytes suppressed the formation of structurally and functionally mature synapses in cortical neuronal cultures. After cuprizone-induced acute demyelination, we observed reduced immunostaining for Nogo-A in the visual cortex accompanied by higher levels of Hevin expression in astrocytes and an increase in excitatory synapse density. Hence, we suggest that interactions between Nogo-A and astrocytes might represent an important pathway of plasticity regulation and could be a target for therapeutic intervention in demyelinating diseases in the future.

The oligodendrocyte growth cone and its actin cytoskeleton: A fundamental element for progenitor cell migration and CNS myelination.

Cells of the oligodendrocyte (OLG) lineage engage in highly motile behaviors that are crucial for effective central nervous system (CNS) myelination. These behaviors include the guided migration of OLG progenitor cells (OPCs), the surveying of local environments by cellular processes extending from differentiating and pre-myelinating OLGs, and during the process of active myelin wrapping, the forward movement of the leading edge of the myelin sheath's inner tongue along the axon. Almost all of these motile behaviors are driven by actin cytoskeletal dynamics initiated within a lamellipodial structure that is located at the tip of cellular OLG/OPC processes and is structurally as well as functionally similar to the neuronal growth cone. Accordingly, coordinated stoichiometries of actin filament (F-actin) assembly and disassembly at these OLG/OPC growth cones have been implicated in directing process outgrowth and guidance, and the initiation of myelination. Nonetheless, the functional importance of the OLG/OPC growth cone still remains to be fully understood, and, as a unique aspect of actin cytoskeletal dynamics, F-actin depolymerization and disassembly start to predominate at the transition from myelination initiation to myelin wrapping. This review provides an overview of the current knowledge about OLG/OPC growth cones, and it proposes a model in which actin cytoskeletal dynamics in OLG/OPC growth cones are a main driver for morphological transformations and motile behaviors. Remarkably, these activities, at least at the later stages of OLG maturation, may be regulated independently from the transcriptional gene expression changes typically associated with CNS myelination.

Glutamate Transporters: Expression and Function in Oligodendrocytes.

Glutamate, the main excitatory neurotransmitter of the vertebrate central nervous system (CNS), is well known as a regulator of neuronal plasticity and neurodevelopment. Such glutamate function is thought to be mediated primarily by signaling through glutamate receptors. Thus, it requires a tight regulation of extracellular glutamate levels and a fine-tuned homeostasis that, when dysregulated, has been associated with a wide range of central pathologies including neuropsychiatric, neurodevelopmental, and neurodegenerative disorders. In the mammalian CNS, extracellular glutamate levels are controlled by a family of sodium-dependent glutamate transporters belonging to the solute carrier family 1 (SLC1) that are also referred to as excitatory amino acid transporters (EAATs). The presumed main function of EAATs has been best described in the context of synaptic transmission where EAATs expressed by astrocytes and neurons effectively regulate extracellular glutamate levels so that synapses can function independently. There is, however, increasing evidence that EAATs are expressed by cells other than astrocytes and neurons, and that they exhibit functions beyond glutamate clearance. In this review, we will focus on the expression and functions of EAATs in the myelinating cells of the CNS, oligodendrocytes. More specifically, we will discuss potential roles of oligodendrocyte-expressed EAATs in contributing to extracellular glutamate homeostasis, and in regulating oligodendrocyte maturation and CNS myelination by exerting signaling functions that have traditionally been associated with glutamate receptors. In addition, we will provide some examples for how dysregulation of oligodendrocyte-expressed EAATs may be involved in the pathophysiology of neurologic diseases.

Sodium-Calcium Exchangers of the SLC8 Family in Oligodendrocytes: Functional Properties in Health and Disease.

The solute carrier 8 (SLC8) family of sodium-calcium exchangers (NCXs) functions as an essential regulatory system that couples opposite fluxes of sodium and calcium ions across plasmalemmal membranes. NCXs, thereby, play key roles in maintaining an ion homeostasis that preserves cellular integrity. Hence, alterations in NCX expression and regulation have been found to lead to ionic imbalances that are often associated with intracellular calcium overload and cell death. On the other hand, intracellular calcium has been identified as a key driver for a multitude of downstream signaling events that are crucial for proper functioning of biological systems, thus highlighting the need for a tightly controlled balance. In the CNS, NCXs have been primarily characterized in the context of synaptic transmission and ischemic brain damage. However, a much broader picture is emerging. NCXs are expressed by virtually all cells of the CNS including oligodendrocytes (OLGs), the cells that generate the myelin sheath. With a growing appreciation of dynamic calcium signals in OLGs, NCXs are becoming increasingly recognized for their crucial roles in shaping OLG function under both physiological and pathophysiological conditions. In order to provide a current update, this review focuses on the importance of NCXs in cells of the OLG lineage. More specifically, it provides a brief introduction into plasmalemmal NCXs and their modes of activity, and it discusses the roles of OLG expressed NCXs in regulating CNS myelination and in contributing to CNS pathologies associated with detrimental effects on OLG lineage cells.

Effects of HIV-1 Tat on oligodendrocyte viability are mediated by CaMKIIß-GSK3ß interactions.

Myelin disruptions are frequently reported in human immunodeficiency virus (HIV)-infected individuals and can occur in the CNS very early in the disease process. Immature oligodendrocytes (OLs) are quite sensitive to toxic increases in [Ca2+ ]i caused by exposure to HIV-1 Tat (transactivator of transcription, a protein essential for HIV replication and gene expression), but sensitivity to Tat-induced [Ca2+ ]i is reduced in mature OLs. Tat exposure also increased the activity of Ca2+ /calmodulin-dependent kinase IIß (CaMKIIß), the major isoform of CaMKII expressed by OLs, in both immature and mature OLs. Since CaMKIIß is reported to interact with glycogen synthase kinase 3ß (GSK3ß), and GSK3ß activity is implicated in OL apoptosis as well as HIV neuropathology, we hypothesized that disparate effects of Tat on OL viability with maturity might be because of an altered balance of CaMKIIß-GSK3ß activities. Tat expression in vivo led to increased CaMKIIß and GSK3ß activity in multiple brain regions in transgenic mice. In vitro, immature murine OLs expressed higher levels of GSK3ß, but much lower levels of CaMKIIß, than did mature OLs. Exogenous Tat up-regulated GSK3ß activity in immature, but not mature, OLs. Tat-induced death of immature OLs was rescued by the GSK3ß inhibitors valproic acid or SB415286, supporting involvement of GSK3ß signaling. Pharmacologically inhibiting CaMKIIß increased GSK3ß activity in Tat-treated OLs, and genetically knocking down CaMKIIß promoted death in mature OL cultures treated with Tat. Together, these results suggest that the effects of Tat on OL viability are dependent on CaMKIIß-GSK3ß interactions, and that increasing CaMKIIß activity is a potential approach for limiting OL/myelin injury with HIV infection.

Inflammatory demyelination alters subcortical visual circuits.

BACKGROUND: Multiple sclerosis (MS) is an inflammatory demyelinating disease classically associated with axonal damage and loss; more recently, however, synaptic changes have been recognized as additional contributing factors. An anatomical area commonly affected in MS is the visual pathway; yet, changes other than those associated with inflammatory demyelination of the optic nerve, i.e., optic neuritis, have not been described in detail. METHODS: Adult mice were subjected to a diet containing cuprizone to mimic certain aspects of inflammatory demyelination as seen in MS. Demyelination and inflammation were assessed by real-time polymerase chain reaction and immunohistochemistry. Synaptic changes associated with inflammatory demyelination in the dorsal lateral geniculate nucleus (dLGN) were determined by immunohistochemistry, Western blot analysis, and electrophysiological field potential recordings. RESULTS: In the cuprizone model, demyelination was observed in retinorecipient regions of the subcortical visual system, in particular the dLGN, where it was found accompanied by microglia activation and astrogliosis. In contrast, anterior parts of the pathway, i.e., the optic nerve and tract, appeared largely unaffected. Under the inflammatory demyelinating conditions, as seen in the dLGN of cuprizone-treated mice, there was an overall decrease in excitatory synaptic inputs from retinal ganglion cells. At the same time, the number of synaptic complexes arising from gamma-aminobutyric acid (GABA)-generating inhibitory neurons was found increased, as were the synapses that contain the N-methyl-D-aspartate receptor (NMDAR) subunit GluN2B and converge onto inhibitory neurons. These synaptic changes were functionally found associated with a shift toward an overall increase in network inhibition. CONCLUSIONS: Using the cuprizone model of inflammatory demyelination, our data reveal a novel form of synaptic (mal)adaption in the CNS that is characterized by a shift of the excitation/inhibition balance toward inhibitory network activity associated with an increase in GABAergic inhibitory synapses and a possible increase in excitatory input onto inhibitory interneurons. In addition, our data recognize the cuprizone model as a suitable tool in which to assess the effects of inflammatory demyelination on subcortical retinorecipient regions of the visual system, such as the dLGN, in the absence of overt optic neuritis.

The Autotaxin-Lysophosphatidic Acid Axis Modulates Histone Acetylation and Gene Expression during Oligodendrocyte Differentiation.

During development, oligodendrocytes (OLGs), the myelinating cells of the CNS, undergo a stepwise progression during which OLG progenitors, specified from neural stem/progenitor cells, differentiate into fully mature myelinating OLGs. This progression along the OLG lineage is characterized by well synchronized changes in morphology and gene expression patterns. The latter have been found to be particularly critical during the early stages of the lineage, and they have been well described to be regulated by epigenetic mechanisms, especially by the activity of the histone deacetylases HDAC1 and HDAC2. The data presented here identify the extracellular factor autotaxin (ATX) as a novel upstream signal modulating HDAC1/2 activity and gene expression in cells of the OLG lineage. Using the zebrafish as an in vivo model system as well as rodent primary OLG cultures, this functional property of ATX was found to be mediated by its lysophospholipase D (lysoPLD) activity, which has been well characterized to generate the lipid signaling molecule lysophosphatidic acid (LPA). More specifically, the lysoPLD activity of ATX was found to modulate HDAC1/2 regulated gene expression during a time window coinciding with the transition from OLG progenitor to early differentiating OLG. In contrast, HDAC1/2 regulated gene expression during the transition from neural stem/progenitor to OLG progenitor appeared unaffected by ATX and its lysoPLD activity. Thus, together, our data suggest that an ATX-LPA-HDAC1/2 axis regulates OLG differentiation specifically during the transition from OLG progenitor to early differentiating OLG and via a molecular mechanism that is evolutionarily conserved from at least zebrafish to rodent. SIGNIFICANCE STATEMENT: The formation of the axon insulating and supporting myelin sheath by differentiating oligodendrocytes (OLGs) in the CNS is considered an essential step during vertebrate development. In addition, loss and/or dysfunction of the myelin sheath has been associated with a variety of neurologic diseases in which repair is limited, despite the presence of progenitor cells with the potential to differentiate into myelinating OLGs. This study characterizes the autotaxin-lysophosphatidic acid signaling axis as a modulator of OLG differentiation in vivo in the developing zebrafish and in vitro in rodent OLGs in culture. These findings provide novel insight into the regulation of developmental myelination, and they are likely to lead to advancing studies related to the stimulation of myelin repair under pathologic conditions.

Oligodendrocytes Are Targets of HIV-1 Tat: NMDA and AMPA Receptor-Mediated Effects on Survival and Development.

Myelin pallor in HIV(+) individuals can occur very early during the disease process. While myelin damage might partly originate from HIV-induced vascular changes, the timing suggests that myelin and/or oligodendrocytes (OLs) may be directly affected. Histological (Golgi-Kopsch, electron microscopy) and biochemical studies have revealed an increased occurrence of abnormal OL/myelin morphology and dysregulated myelin protein expression in transgenic mice expressing the HIV-1 transactivator of transcription (Tat) protein. This suggests that viral proteins by themselves might cause OL injury. Since Tat interacts with NMDARs, we hypothesized that activation of NMDARs and subsequent disruption of cytoplasmic Ca(2+) ([Ca(2+)]i) homeostasis might be one cause of white matter injury after HIV infection. In culture, HIV-1 Tat caused concentration-dependent death of immature OLs, while more mature OLs remained alive but had reduced myelin-like membranes. Tat also induced [Ca(2+)]i increases and Thr-287 autophosphorylation of Ca(2+)/calmodulin-dependent protein kinase II ß (CaMKIIß) in OLs. Tat-induced [Ca(2+)]i was attenuated by the NMDAR antagonist MK801, and also by the AMPA/kainate receptor antagonist CNQX. Importantly, both MK801 and CNQX blocked Tat-induced death of immature OLs, but only MK801 reversed Tat effects on myelin-like membranes. These results suggest that OLs can be direct targets of HIV proteins released from infected cells. Although viability and membrane production are both affected by glutamatergic receptor-mediated Ca(2+) influx, and possibly the ensuing CaMKIIß activation, the roles of AMPARs and NMDARs appear to be different and dependent on the stage of OL differentiation. SIGNIFICANCE STATEMENT: Over 33 million individuals are currently infected by HIV. Among these individuals, ∼60% develop HIV-associated neurocognitive disorders. Myelin damage and white matter injury have been frequently reported in HIV patients but not extensively studied. Clinical studies using combined antiretroviral therapy (cART) together with adjunctive "anti-inflammatory" drugs show no improvement over cART alone, suggesting existence of injury mechanisms in addition to inflammation. In our studies, oligodendrocytes exhibited rapid increases in intracellular Ca(2+) level upon HIV-1 transactivator of transcription (Tat) exposure. Thus, immature and mature oligodendrocytes can be direct targets of Tat. Since ionotropic glutamate receptor antagonists can partially or fully reverse the detrimental effects of Tat, glutamate receptors could be a potential therapeutic target for white matter damage in HIV patients.

CaMKIIß regulates oligodendrocyte maturation and CNS myelination.

CNS myelination and the maturation of the myelinating cells of the CNS, namely oligodendrocytes, are thought to be regulated by molecular mechanisms controlling the actin cytoskeleton. However, the exact nature of these mechanisms is currently only poorly understood. Here we assessed the role of calcium/calmodulin-dependent kinase type II (CaMKII), in particular CaMKIIß, in oligodendrocyte maturation and CNS myelination. Using in vitro culture studies, our data demonstrate that CaMKIIß is critical for the proper morphological maturation of differentiating oligodendrocytes, an aspect of oligodendrocyte maturation that is mediated to a large extent by changes in the cellular cytoskeleton. Furthermore, our data provide evidence for an actin-cytoskeleton-stabilizing role of CaMKIIß in differentiating oligodendrocytes. Using Camk2b knock-out and Camk2b(A303R) mutant mice, our data revealed an in vivo functional role of CaMKIIß in regulating myelin thickness that may be mediated by a non-kinase-catalytic activity. Our data point toward a critical role of CaMKIIß in regulating oligodendrocyte maturation and CNS myelination via an actin-cytoskeleton-regulatory mechanism.

Activation of sodium-dependent glutamate transporters regulates the morphological aspects of oligodendrocyte maturation via signaling through calcium/calmodulin-dependent kinase IIß's actin-binding/-stabilizing domain.

Signaling via the major excitatory amino acid glutamate has been implicated in the regulation of various aspects of the biology of oligodendrocytes, the myelinating cells of the central nervous system (CNS). In this respect, cells of the oligodendrocyte lineage have been described to express a variety of glutamate-responsive transmembrane proteins including sodium-dependent glutamate transporters. The latter have been well characterized to mediate glutamate clearance from the extracellular space. However, there is increasing evidence that they also mediate glutamate-induced intracellular signaling events. Our data presented here show that the activation of oligodendrocyte expressed sodium-dependent glutamate transporters, in particular GLT-1 and GLAST, promotes the morphological aspects of oligodendrocyte maturation. This effect was found to be associated with a transient increase in intracellular calcium levels and a transient phosphorylation event at the serine (S)(371) site of the calcium sensor calcium/calmodulin-dependent kinase type IIß (CaMKIIß). The potential regulatory S(371) site is located within CaMKIIß's previously defined actin-binding/-stabilizing domain, and phosphorylation events within this domain were identified in our studies as a requirement for sodium-dependent glutamate transporter-mediated promotion of oligodendrocyte maturation. Furthermore, our data provide good evidence for a role of these phosphorylation events in mediating detachment of CaMKIIß from filamentous (F)-actin, and hence allowing a remodeling of the oligodendrocyte's actin cytoskeleton. Taken together with our recent findings, which demonstrated a crucial role of CaMKIIß in regulating CNS myelination in vivo, our data strongly suggest that a sodium-dependent glutamate transporter-CaMKIIß-actin cytoskeleton axis plays an important role in the regulation of oligodendrocyte maturation and CNS myelination.

Autotaxin/ENPP2 regulates oligodendrocyte differentiation in vivo in the developing zebrafish hindbrain.

During development, progenitors that are committed to differentiate into oligodendrocytes, the myelinating cells of the central nervous system (CNS), are generated within discrete regions of the neuroepithelium. More specifically, within the developing spinal cord and hindbrain ventrally located progenitor cells that are characterized by the expression of the transcription factor olig2 give temporally rise to first motor neurons and then oligodendrocyte progenitors. The regulation of this temporal neuron-glial switch has been found complex and little is known about the extrinsic factors regulating it. Our studies described here identified a zebrafish ortholog to mammalian atx, which displays evolutionarily conserved expression pattern characteristics. Most interestingly, atx was found to be expressed by cells of the cephalic floor plate during a time period when ventrally-derived oligodendrocyte progenitors arise in the developing hindbrain of the zebrafish. Knock-down of atx expression resulted in a delay and/or inhibition of the timely appearance of oligodendrocyte progenitors and subsequent developmental stages of the oligodendrocyte lineage. This effect of atx knock-down was not accompanied by changes in the number of olig2-positive progenitor cells, the overall morphology of the axonal network or the number of somatic abducens motor neurons. Thus, our studies identified Atx as an extrinsic factor that is likely secreted by cells from the floor plate and that is involved in regulating specifically the progression of olig2-positive progenitor cells into lineage committed oligodendrocyte progenitors.

P2Y12 receptor expression is a critical determinant of functional responsiveness to ATX's MORFO domain.

In the central nervous system, the formation of the myelin sheath and the differentiation of the myelinating cells, namely oligodendrocytes, are regulated by complex signaling networks that involve purinergic receptors and the extracellular matrix. However, the exact nature of the molecular interactions underlying these networks still needs to be defined. In this respect, the data presented here reveal a signaling mechanism that is characterized by an interaction between the purinergic P2Y(12) receptor and the matricellular extracellular matrix protein autotaxin (ATX), also known as ENPP2, phosphodiesterase-Iα/ATX, or lysoPLD. ATX has been previously described by us to mediate intermediate states of oligodendrocyte adhesion and to enable changes in oligodendrocyte morphology that are thought to be crucial for the formation of a fully functional myelin sheath. This functional property of ATX is mediated by ATX's modulator of oligodendrocyte remodeling and focal adhesion organization (MORFO) domain. Here, we show that the expression of the P2Y(12) receptor is necessary for ATX's MORFO domain to exert its effects on differentiating oligodendrocytes. In addition, our data demonstrate that exogenous expression of the P2Y(12) receptor can render cells responsive to the known effects of ATX's MORFO domain, and they identify Rac1 as an intracellular factor mediating the effect of ATX-MORFO-P2Y(12) signaling on the assembly of focal adhesions. Our data further support the idea that a physical interaction between ATX and the P2Y(12) receptor provides the basis for an ATX-MORFO-P2Y(12) signaling axis that is crucial for mediating cellular states of intermediate adhesion and morphological/structural plasticity.

Elucidating the binding mechanism of LPA species and analogs in an LPA4 receptor homology model.

Lysophosphatidic acid receptor 4 (LPA4) has emerged as a potential therapeutic target for the treatment of a variety of diseases, including cancer and obesity-induced diabetes, but its structure remains to be revealed. In the present work, a homology model of LPA4 was built for studying the binding mechanism of LPA species and analogs. Then five selected LPA species and analogs with structural variations in their phosphate groups, substitutions on the glycerol backbone, and fatty acyl chains were docked into the LPA4 model, followed by molecular dynamics simulations and energy analyses. The computational results revealed that the aliphatic residues located at the vertical cleft of LPA4 may form a hydrophobic environment for the fatty acyl moiety of LPA species and their analogs. Meanwhile, the positively charged residues in the central cavity of LPA4 may form ionic interactions with the negatively charged hydrophilic head group of LPA species and their analogs. In addition, it was noted that a different binding mode of the hydrophilic head group in each species with the central cavity of the LPA4 might lead to a special rearrangement of the fatty acyl moiety. Taken together, these results may facilitate understanding of the activation mechanism of LPA4 and help design selective ligands to modulate its function for therapeutic purposes.

Deletion of the Sodium-Dependent Glutamate Transporter GLT-1 in Maturing Oligodendrocytes Attenuates Myelination of Callosal Axons During a Postnatal Phase of Central Nervous System Development.

The sodium-dependent glutamate transporter GLT-1 (EAAT2, SLC1A2) has been well-described as an important regulator of extracellular glutamate homeostasis in the central nervous system (CNS), a function that is performed mainly through its presence on astrocytes. There is, however, increasing evidence for the expression of GLT-1 in CNS cells other than astrocytes and in functional roles that are mediated by mechanisms downstream of glutamate uptake. In this context, GLT-1 expression has been reported for both neurons and oligodendrocytes (OLGs), and neuronal presynaptic presence of GLT-1 has been implicated in the regulation of glutamate uptake, gene expression, and mitochondrial function. Much less is currently known about the functional roles of GLT-1 expressed by OLGs. The data presented here provide first evidence that GLT-1 expressed by maturing OLGs contributes to the modulation of developmental myelination in the CNS. More specifically, using inducible and conditional knockout mice in which GLT-1 was deleted in maturing OLGs during a peak period of myelination (between 2 and 4 weeks of age) revealed hypomyelinated characteristics in the corpus callosum of preferentially male mice. These characteristics included reduced percentages of smaller diameter myelinated axons and reduced myelin thickness. Interestingly, this myelination phenotype was not found to be associated with major changes in myelin gene expression. Taken together, the data presented here demonstrate that GLT-1 expressed by maturing OLGs is involved in the modulation of the morphological aspects associated with CNS myelination in at least the corpus callosum and during a developmental window that appears of particular vulnerability in males compared to females.

Novel forms of neurofascin 155 in the central nervous system: alterations in paranodal disruption models and multiple sclerosis.

Stability of the myelin-axon unit is achieved, at least in part, by specialized paranodal junctions comprised of the neuronal heterocomplex of contactin and contactin-associated protein and the myelin protein neurofascin 155. In multiple sclerosis, normal distribution of these proteins is altered, resulting in the loss of the insulating myelin and consequently causing axonal dysfunction. Previously, this laboratory reported that mice lacking the myelin-enriched lipid sulphatide are characterized by a progressive deterioration of the paranodal structure. Here, it is shown that this deterioration is preceded by significant loss of neurofascin 155 clustering at the myelin paranode. Interestingly, prolonged electrophoretic separation revealed the existence of two neurofascin 155 bands, neurofascin 155 high and neurofascin 155 low, which are readily observed following N-linked deglycosylation. Neurofascin 155 high is observed at 7 days of age and reaches peak expression at one month of age, while neurofascin 155 low is first observed at 14 days of age and constantly increases until 5 months of age. Studies using conditional neurofascin knockout mice indicated that neurofascin 155 high and neurofascin 155 low are products of the neurofascin gene and are exclusively expressed by oligodendrocytes within the central nervous system. Neurofascin 155 high is a myelin paranodal protein while the distribution of neurofascin 155 low remains to be determined. While neurofascin 155 high levels are significantly reduced in the sulphatide null mice at 15 days, 30 days and 4 months of age, neurofascin 155 low levels remain unaltered. Although maintained at normal levels, neurofascin 155 low is incapable of preserving paranodal structure, thus indicating that neurofascin 155 high is required for paranodal stability. Additionally, comparisons between neurofascin 155 high and neurofascin 155 low in human samples revealed a significant alteration, specifically in multiple sclerosis plaques.

Focal adhesion kinase can play unique and opposing roles in regulating the morphology of differentiating oligodendrocytes.

During development cells of the oligodendrocyte lineage undergo significant changes in morphology when they differentiate from migratory oligodendrocyte progenitors, which are mostly bipolar, into post-migratory pre-myelinating oligodendrocytes, which extend complex and expanded process networks, and then finally into mature oligodendrocytes, which generate myelin sheaths required for efficient signal propagation within the nervous system. This extensive morphological remodeling occurs in the context of a complex extracellular environment and requires significant rearrangement of the cell's cytoskeleton. The molecular mechanisms underlying this intricate integration of signals, however, remain poorly understood. A key regulator of extracellular matrix to cytoskeleton signaling is the non-receptor tyrosine kinase FAK (focal adhesion kinase). Here, we report that FAK can regulate the morphology of differentiating post-migratory pre-myelinating oligodendrocytes in a unique and opposing fashion that is dependent on the nature of the extracellular matrix and mediated largely by FAK's catalytic activity. More specifically, FAK was found to restrict process network expansion in the presence of fibronectin but to promote morphological maturation in the presence of laminin-2. In addition, FAK's restraining role predominated for postnatal day 3-derived cells, while its maturation promoting role prevailed for postnatal day 5-derived cells. Taken together, our findings reveal a complex role of FAK in regulating the morphology of post-migratory pre-myelinating oligodendrocytes.

Autotaxin (ATX): a multi-functional and multi-modular protein possessing enzymatic lysoPLD activity and matricellular properties.

Recent studies have established that autotaxin (ATX), also known as phosphodiesterase Ialpha/autotaxin (PD-Ialpha/ATX) or (ecto)nucleotide pyrophosphatase/phosphodiesterase 2 [(E)NPP2], represents a multi-functional and multi-modular protein. ATX was initially thought to function exclusively as a phosphodiesterase/pyrophosphatase. However, it has become apparent that this enzymatically active site, which is ultimately responsible for ATX's originally discovered property of tumor cell motility stimulation, mediates the conversion of lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA). In addition, a separate functionally active domain, here referred to as the Modulator of Oligodendrocyte Remodeling and Focal adhesion Organization (MORFO) domain, was discovered in studies analyzing the role of ATX during the differentiation of myelinating cells of the central nervous system (CNS), namely oligodendrocytes. This novel domain was found to mediate anti-adhesive, i.e. matricellular, properties and to promote morphological maturation of oligodendrocytes. In this review, we summarize our current understanding of ATX's structure-function domains and discuss their contribution to the presently known main functional roles of ATX.

Neurotrophin-3 targets the translational initiation machinery in oligodendrocytes.

Neurotrophin-3 (NT-3) regulates oligodendrocyte (OLG) differentiation by mechanisms that remain poorly understood. Exposure of OLGs to NT-3 induces a significant increase in the levels of myelin basic protein (MBP). However, we found that this stimulation occurs in the absence of measurable effects on MBP gene promoter activation or mRNA expression, suggesting that NT-3 upregulates MBP protein expression by a posttranscriptional mechanism. Furthermore, NT-3 also causes an increase in the levels of myelin-associated glycoprotein (MAG) and myelin OLG glycoprotein (MOG), raising the possibility of a more general effect on myelin protein synthesis. Surprisingly, (35)S-methionine incorporation into total OLG proteins demonstrated a 50% increase in labeling following only a brief, 15-min treatment with NT-3. Such a remarkably fast response is unlikely due to transcriptional activation, reinforcing the possibility that NT-3 may play a crucial role in regulating protein expression by a posttranscriptional mechanism. In support of this idea, we found that NT-3 stimulates the phosphorylation of essential regulators of the initiation machinery, eukaryotic initiation factor 4E (eIF4E), and its inhibitory binding partner 4E binding protein 1 (4EBP1), two crucial players in controlling cap-dependent protein synthesis. This stimulation involves the activation of pathways mediated by ERK1/2 and PI3K/mTOR, implicating these two kinase systems as modulators of protein synthesis in developing OLGs. Altogether, these observations show for the first time that NT-3 has the capacity of targeting the translational machinery and suggest a potential stimulatory effect of this neurotrophin on myelination by direct action on protein translation in the OLGs.

Focal adhesion kinase (FAK): A regulator of CNS myelination.

The formation of the myelin sheath is a crucial step during development because it enables fast and efficient propagation of signals within the limited space of the mammalian central nervous system (CNS). During the process of myelination, oligodendrocytes actively interact with the extracellular matrix (ECM). These interactions are considered crucial for proper and timely completion of the myelin sheath. However, the exact regulatory circuits involved in the signaling events that occur between the ECM and oligodendrocytes are currently not fully understood. Therefore, in the present study we investigated the role of a known integrator of cell-ECM signaling, namely, focal adhesion kinase (FAK), in CNS myelination via the use of conditional (oligodendrocyte-specific) and inducible FAK-knockout mice (Fak(flox/flox): PLP/CreER(T) mice). When inducing FAK knockout just prior to and during active myelination of the optic nerve, we observed a significant reduction in the number of myelinated fibers on postnatal day 14. In addition, our data revealed a decreased number of primary processes extending from oligodendrocyte cell bodies at this postnatal age and on induction of FAK knockout. In contrast, myelination appeared normal on postnatal day 28. Thus, our data suggest that FAK controls the efficiency and timing of CNS myelination during its initial stages, at least in part, by regulating oligodendrocyte process outgrowth and/or remodeling.