Hyaluronan blocks oligodendrocyte progenitor maturation and remyelination through TLR2.

Failure of remyelination is largely responsible for sustained neurologic symptoms in multiple sclerosis (MS). MS lesions contain hyaluronan deposits that inhibit oligodendrocyte precursor cell (OPC) maturation. However, the mechanism behind this inhibition is unclear. We report here that Toll-like receptor 2 (TLR2) is expressed by oligodendrocytes and is up-regulated in MS lesions. Pathogen-derived TLR2 agonists, but not agonists for other TLRs, inhibit OPC maturation in vitro. Hyaluronan-mediated inhibition of OPC maturation requires TLR2 and MyD88, a TLR2 adaptor molecule. Ablated expression of TLR2 also enhances remyelination in a lysolecithin animal model. Hyaluronidases expressed by OPCs degrade hyaluronan to hyaluronan oligomers, a requirement for hyaluronan/TLR2 signaling. MS lesions contain both TLR2(+) oligodendrocytes and low-molecular-weight hyaluronan, consistent with their importance to remyelination in MS. We thus have defined a mechanism controlling remyelination failure in MS where hyaluronan is degraded by hyaluronidases into hyaluronan oligomers that block OPC maturation and remyelination through TLR2-MyD88 signaling.

Sonic hedgehog is required during an early phase of oligodendrocyte development in mammalian brain.

Oligodendrocyte precursor development in the embryonic spinal cord is thought to be regulated by the secreted signal, Sonic hedgehog (Shh). Such precursors can be identified by the expression of Olig genes, encoding basic helix-loop-helix factors, in the spinal cord and brain. However, the signaling pathways that govern oligodendrocyte precursor (OLP) development in the rostral central nervous system are poorly understood. Here, we show that Shh is required for oligodendrocyte development in the mouse forebrain and spinal cord, and that Shh proteins are both necessary and sufficient for OLP production in cortical neuroepithelial cultures. Moreover, adenovirus-mediated Olig1 ectopic expression can promote OLP formation independent of Shh activity. Our results demonstrate essential functions for Shh during early phases of oligodendrocyte development in the mammalian central nervous system. They further suggest that a key role of Shh signaling is activation of Olig genes.

The erbB2 gene is required for the development of terminally differentiated spinal cord oligodendrocytes.

Development of oligodendrocytes and the generation of myelin internodes within the spinal cord depends on regional signals derived from the notochord and axonally derived signals. Neuregulin 1 (NRG)-1, localized in the floor plate as well as in motor and sensory neurons, is necessary for normal oligodendrocyte development. Oligodendrocytes respond to NRGs by activating members of the erbB receptor tyrosine kinase family. Here, we show that erbB2 is not necessary for the early stages of oligodendrocyte precursor development, but is essential for proligodendroblasts to differentiate into galactosylcerebroside-positive (GalC+) oligodendrocytes. In the presence of erbB2, oligodendrocyte development is normal. In the absence of erbB2 (erbB2-/-), however, oligodendrocyte development is halted at the proligodendroblast stage with a >10-fold reduction in the number of GalC+ oligodendrocytes. ErbB2 appears to function in the transition of proligodendroblast to oligodendrocyte by transducing a terminal differentiation signal, since there is no evidence of increased oligodendrocyte death in the absence of erbB2. Furthermore, known survival signals for oligodendrocytes increase oligodendrocyte numbers in the presence of erbB2, but fail to do so in the absence of erbB2. Of the erbB2-/- oligodendrocytes that do differentiate, all fail to ensheath neurites. These data suggest that erbB2 is required for the terminal differentiation of oligodendrocytes and for development of myelin.

Neuregulin-induced association of Sos Ras exchange protein with HER2(erbB2)/HER3(erbB3) receptor complexes in Schwann cells through a specific Grb2-HER2(erbB2) interaction.

Neuregulins are members of the epidermal growth factor family of related ligands that exert pleotropic effects during development on the Schwann cell lineage. The receptor complex activated by neuregulin in Schwann cells consists of HER2 (erbB2) and HER3 (erbB3). The intracellular signaling events that follow activation of the HER2/HER3 receptor complex in primary cells, and in particular in Schwann cells, are poorly understood. We have found that neuregulin induces the rapid association of the guanine nucleotide exchange factor SOS with the HER2/HER3 receptor complex. The association of SOS with the HER2/HER3 receptor complex is preceded by the rapid association of Grb2 with the HER2/HER3 receptor complex. Far Western analysis indicates that Grb2 and SOS bind exclusively to HER2 in the HER2/HER3 receptor complex.

Epidermal growth factor receptor induced apoptosis: potentiation by inhibition of Ras signaling.

Previous studies have shown that certain tumor cell lines which naturally express high levels of the epidermal growth factor receptor (EGFR) undergo apoptosis when exposed to epidermal growth factor. Whether this phenomenon is a direct result of receptor overexpression or some other genetic alteration renders these cells sensitive to apoptosis is yet to be established. We show that experimentally increasing the level of EGFR expression predictably leads to apoptosis in a variety of cell types which requires an active tyrosine kinase but not EGFR autophosphorylation sites. Expression of a dominant negative Ras mutant in EGFR overexpressing cells results in a significant potentiation of EGFR induced apoptosis suggesting that Ras activation is a key survival signal generated by the EGFR. We propose that potentiation of EGFR induced apoptosis by dominant negative Ras results, at least in part, by a block of Akt activation.

The epidermal growth factor receptor engages receptor interacting protein and nuclear factor-kappa B (NF-kappa B)-inducing kinase to activate NF-kappa B. Identification of a novel receptor-tyrosine kinase signalosome.

The transcription factor nuclear factor-kappaB (NF-kappaB) is activated by a diverse number of stimuli including tumor necrosis factor-alpha, interleukin-1, UV irradiation, viruses, as well as receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR). NF-kappaB activation by the tumor necrosis factor receptor (TNFR) involves the formation of a multiprotein complex termed a signalosome. Although previous studies have shown that the activated EGFR can induce NF-kappaB, the mechanism of this activation remains unknown. In this study, we identify components of the signalosome formed by the activated EGFR required to activate NF-kappaB and show that, although the activated EGFR uses mechanisms similar to the TNFR, it recruits a distinct signalosome. We show the EGFR forms a complex with a TNFR-interacting protein (RIP), which plays a key role in TNFR-induced NF-kappaB activation, but not with TRADD, an adaptor protein which serves to recruit RIP to the TNFR. Furthermore, we show that the EGFR associates with NF-kappaB-inducing kinase (NIK) and provide evidence suggesting multiprotein complex formation between the EGFR, RIP, and NIK. Using a dominant negative NIK mutant, we show that NIK activation is required for EGFR-mediated NF-kappaB induction. We also show that a S32/36 IkappaBalpha mutant blocks EGFR-induced NF-kappaB activation. Our studies also suggest that a high level of EGFR expression, a frequent occurrence in human tumors, is optimal for epidermal growth factor-induced NF-kappaB activation. Finally, although protein kinase B/Akt has been implicated in tumor necrosis factor and PDGF-induced NF-kappaB activation, our studies do not support a role for this protein in EGFR-induced NF-kappaB activation.

Growth factor control of CNS myelination.

The molecular signals required for initiating myelination and maintenance of the myelin internode are not known. Several growth factor families have been implicated in promoting oligodendrocyte survival or differentiation and may have consequences on formation of myelin. We developed a reliable assay for detecting ensheathment of neurites by oligodendrocytes in spinal cord explants. This system was used to assay the effect of selected growth factors on myelin internode formation. We examined the influence on myelination of the polypeptide growth factors neuregulin (NRG), platelet-derived growth factor (PDGF), leukemia inhibitory factor (LIF), and the thyroid hormone T(3). We found that NRG, PDGF, and T(3) treatments enhanced myelination while LIF treatment inhibited it. We furthermore found that the most potent combination of factors to enhance myelination was NRG and T(3). Our results demonstrate that the role of growth factors on CNS myelination can be reliably studied in a controlled in vitro environment and that the impact of individual or combinations of growth factors on myelination cannot be predicted by their known effects on oligodendrocyte survival, proliferation, or differentiation.

Neuregulin: an oligodendrocyte growth factor absent in active multiple sclerosis lesions.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) which results in demyelination and axonal injury. Conventional therapy for MS is immune suppression in the absence of agents that promote neural and glial survival or remyelination. Neuregulins are a family of ligands that exert trophic effects on both neurons and glia. Using mice bearing a null mutation in the neuregulin gene, here we demonstrate that neuregulins are necessary for the normal development of oligodendrocytes. In addition, neuregulins are produced in the normal human CNS by astrocytes as well as neurons. Astrocyte-derived neuregulin is functionally active in bioassays and exists in secreted and membrane-associated beta-isoforms. In active and chronic active MS lesions, however, the expression of astrocyte neuregulin is dramatically reduced. The absence of neuregulin in active MS lesions may contribute to the paucity of remyelination in MS.

Neuregulin induces the rapid association of focal adhesion kinase with the erbB2-erbB3 receptor complex in schwann cells.

Neuregulins signal cells by binding to an activating hetero- and homodimeric forms of the neuregulin receptors HER2 (erbB2), HER3 (erbB3), and HER4 (erbB4). Axonally derived neuregulin signals myelin forming cells of the central and peripheral nervous systems through different receptor complexes: oligodendrocytes through erbB2/erbB4 heterodimers and Schwann cells through erbB2/erbB3 heterodimers. Since the leading edge of myelinating cells interacts directly with the axonal surface, we were interested in determining if signaling molecules localized at the leading edge associate with activated neuregulin receptors. We found a novel association between neuregulin receptors and focal adhesion kinase (FAK) in primary cultures of Schwann cells. Following stimulation with ligand, maximal binding of FAK to HER2 occurred by 1 min whereas maximal binding to HER3 was delayed to approximately 7 min. FAK is localized in focal adhesions of Schwann cells. We have previously shown HER2 and HER3 are distributed evenly throughout the plasmalemma. Neuregulins thus use FAK to transmit intracellular signals and the differential kinetics of FAK association with individual neuregulin receptors, as well as its restricted subcellular localization, may play a role in specifying biologic responses.

Identification of the Nogo inhibitor of axon regeneration as a Reticulon protein.

Adult mammalian axon regeneration is generally successful in the peripheral nervous system (PNS) but is dismally poor in the central nervous system (CNS). However, many classes of CNS axons can extend for long distances in peripheral nerve grafts. A comparison of myelin from the CNS and the PNS has revealed that CNS white matter is selectively inhibitory for axonal outgrowth. Several components of CNS white matter, NI35, NI250(Nogo) and MAG, that have inhibitory activity for axon extension have been described. The IN-1 antibody, which recognizes NI35 and NI250(Nogo), allows moderate degrees of axonal regeneration and functional recovery after spinal cord injury. Here we identify Nogo as a member of the Reticulon family, Reticulon 4-A. Nogo is expressed by oligodendrocytes but not by Schwann cells, and associates primarily with the endoplasmic reticulum. A 66-residue lumenal/extracellular domain inhibits axonal extension and collapses dorsal root ganglion growth cones. In contrast to Nogo, Reticulon 1 and 3 are not expressed by oligodendrocytes, and the 66-residue lumenal/extracellular domains from Reticulon 1, 2 and 3 do not inhibit axonal regeneration. These data provide a molecular basis to assess the contribution of Nogo to the failure of axonal regeneration in the adult CNS.

Failure of spinal cord oligodendrocyte development in mice lacking neuregulin.

Oligodendrocytes develop from a subpopulation of precursor cells within the ventral ventricular zone of the spinal cord. The molecular cues that direct this spatially and temporally restricted event seem to originate in part from structures ventral to and within the spinal cord. Here, we present evidence that the family of ligands termed neuregulins are necessary for the normal generation of mouse spinal cord oligodendrocytes. Oligodendrocytes mature in spinal cord explants from wild-type mice and mice heterozygotic for a null mutation in the neuregulin gene (NRG +/-) in a temporal sequence of developmental events that replicates that observed in vivo. However, in spinal cord explants derived from mice lacking neuregulin (NRG -/-), oligodendrocytes fail to develop. Addition of recombinant neuregulin to spinal cord explants from NRG -/- mice rescues oligodendrocyte development. In wild-type spinal cord explants, inhibitors of neuregulin mimic the inhibition of oligodendrocyte development that occurs in NRG -/- explants. In embryonic mouse spinal cord, neuregulins are present in motor neurons and the ventral ventricular zone where they likely exert their influence on early oligodendrocyte precursor cells.

Axonal neuregulin signals cells of the oligodendrocyte lineage through activation of HER4 and Schwann cells through HER2 and HER3.

We are interested in the signaling between axons and glia that leads to myelination and maintenance of the myelin internode, and we have focused on the role of neuregulins and their receptors. Neuregulins are a family of ligands that includes heregulin, neu differentiation factor, glial growth factor, and the acetylcholine receptor-inducing activity. Three signal transducing transmembrane receptors for neuregulins, which bear significant homology to the EGF receptor, are currently known: HER2 (erbB2), HER3 (erbB3), and HER4 (erbB4). We have found that oligodendrocite-type II astrocyte (O2A) progenitor cells and mature oligodendrocytes express HER2 and HER4 but no HER3. Schwann cells express HER2 and HER3 but little HER4. In O2A progenitor cells and oligodendrocytes, recombinant neuregulin induces the rapid tyrosine phosphorylation of only HER4. HER2 is not phosphorylated in cells of the oligodendrocyte lineage, but a physical interaction between HER2 and HER4 was detected in coimmunoprecipitation experiments. In Schwann cells, neuregulin induces the phosphorylation of both HER2 and HER3. Coimmunoprecipitation experiments indicate that receptor activation in Schwann cells results in the formation of HER2:HER3 heterodimers. Neuregulin localized immunocytochemically was present on neurites of cultured dorsal root ganglion neurons, and it was released into the medium in a form that promoted receptor tyrosine phosphorylation. Neuregulins therefore meet important criteria expected of molecules involved in axonal-glial signaling. The use of unique neuregulin receptor combinations in oligodendrocytes and Schwann cells likely results in recruitment of different signaling pathways and thus provides a basis for different biological responses.

Interferon-gamma-induced oligodendrocyte cell death: implications for the pathogenesis of multiple sclerosis.

BACKGROUND: The histopathology of multiple sclerosis (MS) is characterized by a loss of myelin and oligodendrocytes, relative preservation of axons, and a modest inflammatory response. The reasons for this selective oligodendrocyte death and demyelination are unknown. MATERIALS AND METHODS: In light of the T lymphocyte and macrophage infiltrates in MS lesions and the numerous cytokines these cells secrete, the direct influence of cytokines on survival of cultured oligodendrocytes and sensory neurons was investigated. Expression of cytokines in vivo was determined by immunolabeling cryostat sections of snap-frozen tissue containing chronic active lesions from four different patients. The samples were also analyzed for the presence of apoptotic nuclei by in situ labeling of 3'-OH ends of degraded nuclear DNA. RESULTS: The results showed: (i) interferon-gamma (IFN gamma) to be a potent inducer of apoptosis among oligodendrocytes in vitro and that this effect can be reversed by leukemia inhibitory factor (LIF); (ii) IFN gamma has a minimal effect on the survival of cultured neurons; (iii) IFN gamma at the margins of active MS plaques but not in unaffected white matter; (iv) evidence for apoptosis of oligodendrocytes at the advancing margins of chronic active MS plaques. CONCLUSIONS: Injury to a substantial number of oligodendrocytes in MS is the results of programmed cell death rather than necrotic cell death mechanisms. We postulate that IFN gamma plays a role in the pathogenesis of MS by activating apoptosis in oligodendrocytes.

A role for the acetylcholine receptor-inducing protein ARIA in oligodendrocyte development.

ARIA acetylcholine receptor-inducing activity protein, is a member of a family of ligands that includes the Neu differentiation factor, heregulin, and glial growth factor. These ligands all act through one or more receptor tyrosine kinases of approximately 185 kDa. In some conditions these ligands promote proliferation, whereas in others they induce differentiation. ARIA was originally isolated from chick brain on the basis of its ability to induce synthesis of nicotinic acetylcholine receptors in skeletal muscle. In this paper we show that ARIA is expressed in the subventricular zone of the rat brain and that it enhances the development of oligodendrocytes from bipotential (O2A) glial progenitor cells. We have also found that ARIA induces tyrosine phosphorylation of a 185-kDa protein in O2A progenitor cells. ARIA does not increase bromodeoxyuridine incorporation by oligodendrocytes but is mitogenic when added to Schwann cells in vitro. Thus, ARIA accelerates the formation of oligodendrocytes in vitro and is expressed where it could exercise the same influence in vivo.

Is multiple sclerosis an autoimmune disease?

It is a commonly held view that multiple sclerosis (MS) may be an autoimmune disease. Most neurology texts list MS as an autoimmune disease and most texts on autoimmunity point to MS as a prime example of an autoimmune disease of the CNS. This view has influenced research into the pathogenesis of MS to the extent that most published work on MS is based on the assumption that it is correct, ignoring other possibilities, unless they can be put into an autoimmune context. Furthermore, most attempts at treating MS have been with agents that influence the immune system. Some of these agents, such as cytoxan and cyclosporin, are drugs with serious side-effects. Hence, it can be argued that over the years some MS patients have suffered because of the autoimmune hypothesis, although in the end and even today, other MS patients may benefit from it. This article examines some of the evidence in support of autoimmune hypotheses of the pathogenesis of MS. We believe that this examination shows that it has not been proven that MS is an autoimmune disease although it underscores the possibility that it may be so. Although it is still a reasonable hypothesis that MS is an autoimmune disease, it has yet to be proven and it would constitute a serious error of omission not to examine other possibilities.

Mediation by G proteins of signals that cause collapse of growth cones.

During development, motion of nerve growth cones ceases on contact with particular targets. The signaling mechanism is unknown. In culture, growth cone collapse can be caused by solubilized embryonic brain membranes, central nervous system myelin, a 35-kilodalton protein isolated from myelin, and mastoparan. Collapse induced by each of these is blocked by pertussis toxin. Thus, collapse of growth cones is mediated by G protein-coupled receptors, which may be activated by proteins associated with the cell surface as well as by soluble ligands.

GAP-43 as a plasticity protein in neuronal form and repair.

Neurons exhibit a remarkable plasticity of form, both during neural development and during the subsequent remodelling of synaptic connectivity. Here we review work on GAP-43 and G0, and focus upon the thesis that their interaction may endow neurons with such plasticity. We also present new data on the role of G proteins in neurite growth, and on the interaction of GAP-43 and actin. GAP-43 is a protein induced during periods of axonal extension and highly enriched on the inner surface of the growth cone membrane. Its membrane localization is primarily due to a short amino terminal sequence which is subject to palmitoylation. Binding to actin filaments may also assist in restricting the protein to specific cellular domains. Consistent with its role as a "plasticity protein," there is evidence that GAP-43 can directly alter cell shape and neurite extension, and several theses have been advanced for how it might do so. Two other prominent components of the growth cone membrane are the alpha and beta subunits of G0. GAP-43 regulates their guanine nucleotide exchange, which is an unusual role for an intracellular protein. We speculate that GAP-43 may adjust the "set point" of responsiveness for G0 stimulation by receptors, thereby altering the neuronal propensity to growth, without actually causing growth. To begin to address how G protein activity affects axon growth, we have developed a means to introduce guanine nucleotide analogs into sympathetic neurons. Stimulation of G proteins with GTP-gamma-S retards axon growth, whereas GDP-beta-S enhances it. This is compatible with G protein registration of inhibitory signals.

Oligodendrocyte-substratum adhesion activates the synthesis of specific lipid species involved in cell signaling.

Ovine oligodendrocytes (OLGs) undergo biochemical and morphological changes following attachment to polylysine. Autoradiographs of two-dimensional thin-layer chromatograms of [14C]Gal-labeled OLG cultures revealed that attachment of OLGs to a polylysine substratum and their subsequent morphological differentiation is accompanied by an increased synthesis of multiple forms of galactosylceramide, sulfogalactosylceramide, and both sulfogalactosyl- and galactosyl-diglycerides, together with an array of complex sialoglycosphingolipids, predominantly GM2 ganglioside. As previously reported, overall lipid synthesis measured by [14C]acetate incorporation into glycerophosphatides, sphingomyelin, and neutral lipids also increased dramatically for up to 60 days (last time point examined) following OLG-substratum adhesion, reflecting membrane growth. Attachment was associated with a rapid augmentation in the synthesis of ethanolamine plasmalogen from 12 to 27% within 24 hr to reach a 35% plateau at 30 days and remain constant thereafter. In contrast, the plasmalogen content of phosphatidylcholine remained constant at 3-5%. This rapid increase in lipid synthesis (especially in the ethanolamine plasmalogen content following attachment) closely paralleled increased diacylglycerol (DAG) production and protein kinase C-dependent phosphorylation of both myelin basic protein and 2',3'-cyclic nucleotide phosphohydrolase. Labeling studies indicated that the major source of [3H]arachidonate-labeled DAG following attachment was from phosphatidylinositol turnover (and to a lesser extent phosphatidylcholine) rather than polyphosphoinositides or plasmalogens. Enhanced lipid synthesis is not only required for the production of membranes in these myelin-producing cells but is also a source of second messengers required in the posttranslational modification of key myelin and cellular proteins.

Growth cone transduction: Go and GAP-43.

The neuronal growth cone plays a crucial role in forming the complex brain architecture achieved during development, and similar nerve terminal mechanisms may operate to modify synaptic structure during adulthood. The growth cone leads the elongating axon towards appropriate synaptic targets by altering motility in response to a variety of extracellular signals. Independently of extrinsic clues, neurons manifest intrinsic control of their growth and form (Banker and Cowan, 1979). Hence, there must be intracellular proteins which control nerve cell shape, so-called 'plasticity' or 'growth' genes. GAP-43 may be such a molecule (Skene and Willard, 1981; Benowitz and Lewis, 1983). For example, GAP-43 is localized to the growth cone membrane (Meiri et al. 1986; Skene et al. 1986) and can enhance filopodial formation even in non-neuronal cells (Zuber et al. 1989a). It includes a small region at the amino terminus for membrane association and perhaps growth cone targeting (Zuber et al. 1989b, Liu et al. 1991). We have found that Go, a member of the G protein family that links receptors and second messengers, is the major non-cytoskeletal protein in the growth cone membrane (Strittmatter et al. 1990). Double staining immunohistochemistry for GAP-43 and Go shows that the distributions of the two proteins are quite similar. Purified GAP-43 regulates the activity of purified Go (Strittmatter et al. 1990), a surprising observation since GAP-43 is an intracellular protein. We have compared the mechanism of GAP-43 activation of Go with that of G protein-linked receptors.2+ interactions between Go and GAP-43 suggest that Go plays a pivotal role in growth cone function, coordinating the effects of both extracellular signals and intracellular growth proteins.