F3/contactin acts as a functional ligand for Notch during oligodendrocyte maturation

Axon-derived molecules are temporally and spatially required as positive or negative signals to coordinate oligodendrocyte differentiation. Increasing evidence suggests that, in addition to the inhibitory Jagged1/Notch1 signaling cascade, other pathways act via Notch to mediate oligodendrocyte differentiation. The GPI-linked neural cell recognition molecule F3/contactin is clustered during development at the paranodal region, a vital site for axoglial interaction. Here, we show that F3/contactin acts as a functional ligand of Notch. This trans-extracellular interaction triggers gamma-secretase-dependent nuclear translocation of the Notch intracellular domain. F3/Notch signaling promotes oligodendrocyte precursor cell differentiation and upregulates the myelin-related protein MAG in OLN-93 cells. This can be blocked by dominant negative Notch1, Notch2, and two Deltex1 mutants lacking the RING-H2 finger motif, but not by dominant-negative RBP-J or Hes1 antisense oligonucleotides. Expression of constitutively active Notch1 or Notch2 does not upregulate MAG. Thus, F3/contactin specifically initiates a Notch/Deltex1 signaling pathway that promotes oligodendrocyte maturation and myelination.     

Multiple sclerosis: re-expression of a developmental pathway that restricts oligodendrocyte maturation

During mammalian central nervous system (CNS) development, contact-mediated activation of Notch1 receptors on oligodendrocyte precursors by the ligand Jagged1 induces Hes5, which inhibits maturation of these cells. Here we tested whether the Notch pathway is re-expressed in the adult CNS in multiple sclerosis (MS), an inflammatory demyelinating disease in which remyelination is typically limited. We found that transforming growth factor-beta 1 (TGF-beta 1), a cytokine upregulated in MS, specifically re-induced Jagged1 in primary cultures of human astrocytes. Within and around active MS plaques lacking remyelination, Jagged1 was expressed at high levels by hypertrophic astrocytes, whereas Notch1 and Hes5 localized to cells with an immature oligodendrocyte phenotype, and TGF-beta 1 was associated with perivascular extracellular matrix in the same areas. In contrast, there was negligible Jagged1 expression in remyelinated lesions. Experiments in vitro showed that Jagged1 signaling inhibited process outgrowth from primary human oligodendrocytes. These data are the first to implicate the Notch pathway in the limited remyelination in MS. Thus, Notch may represent a potential target for therapeutic intervention in this disease.     

Contact with central nervous system myelin inhibits oligodendrocyte progenitor maturation

Oligodendrocytes, the myelinating cells of the central nervous system (CNS), are generated during development through the proliferation and differentiation of a distinct progenitor population. Not all oligodendrocyte progenitors generated during development differentiate, however, and large numbers of oligodendrocyte progenitors are present in the adult CNS, particularly in white matter. These "adult progenitors" can be identified through expression of the NG2 proteoglycan. Adult oligodendrocyte progenitors are thought to develop from the original pool of progenitors and in vitro are capable of differentiating into oligodendrocytes. Why these cells fail to differentiate in the intact CNS is currently unclear. Here we show that contact with CNS myelin inhibits the maturation of immature oligodendrocyte progenitors. The inhibition of oligodendrocyte progenitor maturation is a characteristic of CNS myelin that is not shared by several other membrane preparations including adult and neonatal neural membrane fractions, PNS myelin, or liver. This inhibition is concentration dependent, is reversible, and appears not to be mediated by either myelin basic protein or basic fibroblast growth factor. Myelin-induced inhibition of oligodendrocyte progenitor maturation provides a mechanism to explain the generation of a residual pool of immature oligodendrocyte progenitors in the mature CNS.     

Hyperforin promotes mitochondrial function and development of oligodendrocytes

St. John's wort has been found to be an effective and safe herbal treatment for depression in several clinical trials. However, the underlying mechanism of its therapeutic effects is unclear. Recent studies show that the loss and malfunction of oligodendrocytes are closely related to the neuropathological changes in depression, which can be reversed by antidepressant treatment. In this study, we evaluated the effects of hyperforin, a major active component of St. John's wort, on the proliferation, development and mitochondrial function of oligodendrocytes. The study results revealed that hyperforin promotes maturation of oligodendrocytes and increases mitochondrial function without affecting proliferation of an oligodendrocyte progenitor cell line and neural stem/progenitor cells. Hyperforin also prevented mitochondrial toxin-induced cytotoxicity in an oligodendrocyte progenitor cell line. These findings suggest that hyperforin may stimulate the development and function of oligodendrocytes, which could be a mechanism of its effect in depression. Future in vitro and in vivo studies are required to further characterize the mechanisms of hyperforin.     

Delta-Notch signaling regulates oligodendrocyte specification

Oligodendrocytes, the myelinating cell type of the central nervous system, arise from a ventral population of precursors that also produces motoneurons. Although the mechanisms that specify motoneuron development are well described, the mechanisms that generate oligodendrocytes from the same precursor population are largely unknown. By analysing mutant zebrafish embryos, we found that Delta-Notch signaling is required for spinal cord oligodendrocyte specification. Using a transgenic, conditional expression system, we also learned that constitutive Notch activity could promote formation of excess oligodendrocyte progenitor cells (OPCs). However, excess OPCs are induced only in ventral spinal cord at the time that OPCs normally develop. Our data provide evidence that Notch signaling maintains subsets of ventral spinal cord precursors during neuronal birth and, acting with other temporally and spatially restricted factors, specifies them for oligodendrocyte fate.     

A method for deriving homogenous population of oligodendrocytes from mouse embryonic stem cells

There is a pressing need for new therapeutics for the generation and transplantation of oligodendrocyte to the white matter to help replace and render injured cells that are lost in demyelinating disease. There are a few protocols describing a homogenous derivation of non-manipulated mouse embryonic stem cells to oligodendrocytes (ES-OL). Moreover, protocols that are successful in producing ES-OL do so with low efficiency. Therefore, we describe clear methodology for differentiation of mouse ES cells to oligodendrocyte to a high degree of homogenity and reproducibility in vitro. In addition, taking advantage of three defined media, we can generate a defined ES to oligodendrocyte lineage while selecting against neurons and astrocytes. More specifically, (1) Glial stem cell defining media (GSCDM), supplemented with appropriate combination of SHH and RA support pro-oligodendrocyte developing neural spheres from ES cells, (2) Oligodendrocyte differentiating media, induces lineage selection of oligodendrocytes progenitors from neural stem cells, and (3) Oligodendrocyte maturation media, supports oligodendrocytes progenitor maturation. Moreover, the ES cell derived oligodendrocytes display mature properites in the prescence of rat dorsal root gangila in vitro. Thus confirming thier potential for use to invesitgate developmental pathways and future potential use of cells in transplantation towards myelin repair.     

Notch signaling: a rheostat regulating oligodendrocyte differentiation?

Recent studies suggest that Notch signaling provides both instructive and inhibitory cues for oligodendroglial differentiation, depending on the developmental stage and the stimulatory ligand. In the October 17 issue of Cell, Hu et al. present the axonal cell adhesion molecule contactin as a functional Notch ligand, and suggest interesting potential roles for axoglial interactions in regulating oligodendroglial maturation.     

Compartmentation of Fyn kinase with glycosylphosphatidylinositol-anchored molecules in oligodendrocytes facilitates kinase activation during myelination.

In many cell types, glycosylphosphatidylinositol (GPI)-anchored proteins are sequestered in detergent-resistant membrane rafts. These are plasma membrane microdomains enriched in glycosphingolipids and cholesterol and are suggested to be platforms for cell signaling. Concomitant with the synthesis of myelin glycosphingolipids, maturing oligodendrocytes progressively associate GPI-anchored proteins, including the adhesion molecules NCAM 120 and F3, in rafts. Here we show that these microdomains include Fyn and Lyn kinases. Both kinases are maximally active in myelin prepared from young animals, correlating with early stages of myelination. In the rafts, Fyn kinase is tightly associated with NCAM 120 and F3. In contrast, in oligodendrocyte progenitor cells lacking rafts or in raft-free membrane domains of more mature cells, F3 does not associate with Fyn. The addition of anti-F3 antibodies to oligodendrocytes results in stimulation of Fyn kinase specifically in rafts. Compartmentation of oligodendrocyte GPI-anchored proteins in rafts is thus a prerequisite for association with Fyn, permitting kinase activation. Interaction of oligodendrocyte F3 with axonal ligands such as L1 and ensuing kinase activation may play a crucial role in initiating myelination.     

Loss of testicular orphan receptor 4 impairs normal myelination in mouse forebrain

Testicular orphan nuclear receptor 4 (TR4) has been suggested to play important roles in the development and functioning of the central nervous system (CNS). We find reduced myelination in TR4 knockout (TR4(-/-)) mice, which is particularly obvious in forebrains and in early developmental stages. Further analysis reveals that CC-1-positive (CC-1+) oligodendrocytes are decreased in TR4(-/-) forebrains. The O4+ signals are also reduced in TR4(-/-) forebrains when examined at postnatal d 7. However, the number and proliferation rate of platelet-derived growth factor receptor alpha-positive (PDGFalphaR+) oligodendrocyte precursor cells (OPCs) remain unaffected in these regions, suggesting that loss of TR4 interrupts oligodendrocyte differentiation. This is further supported by the observation that CC-1+ oligodendrocytes derived from 5-bromo-2'-deoxyuridine incorporating OPCs are significantly reduced in TR4(-/-) forebrains. We also find higher Jagged1 expression levels in axon fiber-enriched regions in TR4(-/-) forebrains, suggesting a more activated Notch signaling in these regions that correlates with previous reports showing that Notch activation inhibits oligodendrocyte differentiation. Together, our results suggest that TR4 is required for proper myelination in the CNS and is particularly important for oligodendrocyte differentiation and maturation in the forebrain regions. The altered Jagged1-Notch signaling in TR4(-/-) forebrain underlies a potential mechanism that contributes to the reduced myelination in the forebrain.     

Neural recognition molecules CHL1 and NB-3 regulate apical dendrite orientation in the neocortex via PTP alpha

Apical dendrites of pyramidal neurons in the neocortex have a stereotypic orientation that is important for neuronal function. Neural recognition molecule Close Homolog of L1 (CHL1) has been shown to regulate oriented growth of apical dendrites in the mouse caudal cortex. Here we show that CHL1 directly associates with NB-3, a member of the F3/contactin family of neural recognition molecules, and enhances its cell surface expression. Similar to CHL1, NB-3 exhibits high-caudal to low-rostral expression in the deep layer neurons of the neocortex. NB-3-deficient mice show abnormal apical dendrite projections of deep layer pyramidal neurons in the visual cortex. Both CHL1 and NB-3 interact with protein tyrosine phosphatase alpha (PTPalpha) and regulate its activity. Moreover, deep layer pyramidal neurons of PTPalpha-deficient mice develop misoriented, even inverted, apical dendrites. We propose a signaling complex in which PTPalpha mediates CHL1 and NB-3-regulated apical dendrite projection in the developing caudal cortex.     

Adropin Is a Brain Membrane-bound Protein Regulating Physical Activity via the NB-3/Notch Signaling Pathway in Mice

Adropin is a highly conserved polypeptide that has been suggested to act as an endocrine factor that plays important roles in metabolic regulation, insulin sensitivity, and endothelial functions. However, in this study, we provide evidence demonstrating that adropin is a plasma membrane protein expressed abundantly in the brain. Using a yeast two-hybrid screening approach, we identified NB-3/Contactin 6, a brain-specific, non-canonical, membrane-tethered Notch1 ligand, as an interaction partner of adropin. Furthermore, this interaction promotes NB3-induced activation of Notch signaling and the expression of Notch target genes. We also generated and characterized adropin knockout mice to explore the role of adropin in vivo. Adropin knockout mice exhibited decreased locomotor activity and impaired motor coordination coupled with defective synapse formation, a phenotype similar to NB-3 knockout mice. Taken together, our data suggest that adropin is a membrane-bound protein that interacts with the brain-specific Notch1 ligand NB3. It regulates physical activity and motor coordination via the NB-3/Notch signaling pathway and plays an important role in cerebellum development in mice.     

PDGF receptors on cells of the oligodendrocyte-type-2 astrocyte (O-2A) cell lineage

It has been shown previously that cultures of rat optic nerve contain three types of macroglial cells--oligodendrocytes and two types of astrocytes. Type-1 astrocytes develop from their own precursor cells beginning before birth, while oligodendrocytes and type-2 astrocytes develop postnatally from a common bipotential precursor called the O-2A progenitor cell. Proliferating O-2A progenitor cells give rise to postmitotic oligodendrocytes beginning around birth, and to type-2 astrocytes beginning in the second postnatal week. Studies in vitro have suggested that platelet-derived growth factor (PDGF), secreted by type-1 astrocytes, plays an important part in timing oligodendrocyte development: PDGF seems to keep O-2A progenitor cells proliferating until an intrinsic clock in the progenitor cells initiates the process leading to oligodendrocyte differentiation. The clock apparently determines when a progenitor cell becomes unresponsive to PDGF, at which point the cell stops dividing and, as a consequence, automatically differentiates into an oligodendrocyte. Here we have used radiolabelled PDGF to show that O-2A progenitor cells have PDGF receptors, suggesting that these cells respond directly to PDGF. The receptors resemble the type A PDGF receptor previously described on human fibroblasts and are initially retained when progenitor cells stop dividing and develop in vitro into oligodendrocytes. The latter finding indicates that receptor loss is not the reason that progenitor cells initially become mitotically unresponsive to PDGF.     

Constitutive expression of interleukin-1beta (IL-1beta) in rat oligodendrocytes

The RT-PCR analysis of RNA from progenitor and differentiated primary rat oligodendrocytes, and from the oligodendrocyte CG-4 cell line, shows the presence of the IL-1beta mRNA, the type I IL-1beta receptor and the IL-1 receptor accessory protein in these cells. In situ hybridization of a rat IL-1beta probe to primary progenitor and differentiated rat oligodendrocytes results in a positive signal. The double hybridization of the IL-1beta probe, together with an oligodendrocyte-specific differentiation marker, to sections of postnatal rat brain at different stages of differentiation is also positive. The double immuno-labelling technique utilized indicates coincidence of the signals on the brain slices. The results show that IL-1beta mRNA is constitutively expressed in rat brain oligodendrocytes from 1 day after birth onward. In agreement with this observation, CG-4 cells, primary progenitor and differentiated rat oligodendrocytes are positively stained by antibodies against IL-1beta. Postnatal brain slices from 1 and 4 day old and adult rats, labelled with a double immunofluorescence technique, are also stained by antibodies against IL-1beta. This signal coincides with that of antibodies against oligodendrocyte-specific surface markers. We conclude that IL-1beta is constitutively expressed in rat brain progenitor and differentiated oligodendrocytes.     

Abnormal myelination in the spinal cord of PTPα-knockout mice

PTPα interacts with F3/contactin to form a membrane-spanning co-receptor complex to transduce extracellular signals to Fyn tyrosine kinase. As both F3 and Fyn regulate myelination, we investigated a role for PTPα in this process. Here, we report that both oligodendrocytes and neurons express PTPα that evenly distributes along myelinated axons of the spinal cord. The ablation of PTPα in vivo leads to early formation of transverse bands that are mainly constituted by F3 and Caspr along the axoglial interface. Notably, PTPα deficiency facilitates abnormal myelination and pronouncedly increases the number of non-landed oligodendrocyte loops at shortened paranodes in the spinal cord. Small axons, which are normally less myelinated, have thick myelin sheaths in the spinal cord of PTPα-null animals. Thus, PTPα may be involved in the formation of axoglial junctions and ensheathment in small axons during myelination of the spinal cord.     

Contactins: Emerging key roles in the development and function of the nervous system

Contactins are a subgroup of molecules belonging to the immunoglobulin superfamily that are expressed exclusively in the nervous system. The subgroup consists of six members: contactin, TAG-1, BIG-1, BIG-2, NB-2 and NB-3. Since their identification in the late 1980s, contactin and TAG-1 have been studied extensively. Axonal expression and the neurite extension activity of contactin and TAG-1 attracted researchers to study the function of these molecules in axon guidance during development. After the exciting discovery of the molecular function of contactin and TAG-1 in myelination earlier this decade, these two molecules have come to be known as the principal molecules in the function and maintenance of myelinated neurons. In contrast, the function of the other four members of this subgroup remained unknown until recently. Here, we will give an overview of contactin function, including recent progress on BIG-2, NB-2 and NB-3.Key words: contactin, GPI-anchor, nervous system, development, cerebellum, myelin, synapse, psychiatric disorder