CD44 enhances neuregulin signaling by Schwann cells

We describe a key role for the CD44 transmembrane glycoprotein in Schwann cell-neuron interactions. CD44 proteins have been implicated in cell adhesion and in the presentation of growth factors to high affinity receptors. We observed high CD44 expression in early rat neonatal nerves at times when Schwann cells proliferate but low expression in adult nerves, where CD44 was found in some nonmyelinating Schwann cells and to varying extents in some myelinating fibers. CD44 constitutively associated with erbB2 and erbB3, receptor tyrosine kinases that heterodimerize and signal in Schwann cells in response to neuregulins. Moreover, CD44 significantly enhanced neuregulin-induced erbB2 phosphorylation and erbB2-erbB3 heterodimerization. Reduction of CD44 expression in vitro resulted in loss of Schwann cell-neurite adhesion and Schwann cell apoptosis. CD44 is therefore crucial for maintaining neuron-Schwann cell interactions at least partly by facilitating neuregulin-induced erbB2-erbB3 activation.     

Membrane-bound neuregulin1 type III actively promotes Schwann cell differentiation of multipotent Progenitor cells

Many steps of peripheral glia development appear to be regulated by neuregulin1 (NRG1) signaling but the exact roles of the different NRG1 isoforms in these processes remain to be determined. While glial growth factor 2 (GGF2), a NRG1 type II isoform, is able to induce a satellite glial fate in neural crest stem cells, targeted mutations in mice have revealed a prominent role of NRG1 type III isoforms in supporting survival of Schwann cells at early developmental stages. Here, we investigated the role of NRG1 isoforms in the differentiation of Schwann cells from neural crest-derived progenitor cells. In multipotent cells isolated from dorsal root ganglia, soluble NRG1 isoforms do not promote Schwann cell features, whereas signaling by membrane-associated NRG1 type III induces the expression of the Schwann cell markers Oct-6/SCIP and S100 in neighboring cells, independent of survival. Thus, axon-bound NRG1 might actively promote both Schwann cell survival and differentiation.     

Neuregulins in Glial Cells

The role of growth factors in controlling the development of glial cells in both the peripheral and central nervous systems has been investigated for a number of years. The recent discovery of a new family of growth factors termed the neuregulins (NRGs) has led to an explosion of information concerning the putative role of these growth factors in the development of Schwann cells (SC), oligodendrocytes (OLG), and astrocytes. Many of these previous studies have focused on the effects of exogenous NRGs on glial cell development and differentiation. We now review the evidence that these glial cells themselves produce NRGs and discuss the major implications of these findings with respect to glial cell development and diseases which affect glial cell function. We also discuss the potential role of endogenous NRGs following neural injury.     

Synergistic interactions between transforming growth factor beta and fibroblast growth factor regulate Schwann cell mitosis

Cultured Schwann cells divide in response to a limited repertoire of mitogens. In addition to cyclic AMP analogs and reagents that raise intracellular cyclic AMP, the only purified mitogens for Schwann cells are transforming growth factor beta (TGFβ), acidic (a) and basic (b) fibroblast growth factor (FGF), and the BB and AB dimers of platelet-derived growth factor (PDGF). Although individually each one of these growth factors is only weakly mitogenic, it is shown here that when TGFβ and bFGF are added to Schwann cell cultures together, they interact to produce a mitogenic response that is much greater than that produced by either growth factor alone. Both the absolute concentration of each protein and the molar ratio of TGFβ to bFGF determines the magnitude of the Schwann cell response.     

In vivo knockdown of ErbB3 in mice inhibits Schwann cell precursor migration

The myelin sheath insulates neuronal axons and markedly increases the nerve conduction velocity. In the peripheral nervous system (PNS), Schwann cell precursors migrate along embryonic neuronal axons to their final destinations, where they eventually wrap around individual axons to form the myelin sheath after birth. ErbB2 and ErbB3 tyrosine kinase receptors form a heterodimer and are extensively expressed in Schwann lineage cells. ErbB2/3 is thought to be one of the primary regulators controlling the entire Schwann cell development. ErbB3 is the bona fide Schwann cell receptor for the neuronal ligand neuregulin-1. Although ErbB2/3 is well known to regulate both Schwann cell precursor migration and myelination by Schwann cells in fishes, it still remains unclear whether in mammals, ErbB2/3 actually regulates Schwann cell precursor migration. Here, we show that knockdown of ErbB3 using a Schwann cell-specific promoter in mice causes delayed migration of Schwann cell precursors. In contrast, littermate control mice display normal migration. Similar results are seen in an in vitro migration assay using reaggregated Schwann cell precursors. Also, ErbB3 knockdown in mice reduces myelin thickness in sciatic nerves, consistent with the established role of ErbB3 in myelination. Thus, ErbB3 plays a key role in migration, as well as in myelination, in mouse Schwann lineage cells, presenting a genetically conservative role of ErbB3 in Schwann cell precursor migration.     

Oriented Schwann cell growth on microgrooved surfaces

Silicon wafers bearing microgrooved surfaces with various groove width, spacing, and depth were fabricated using microlithography. The orientation of rat Schwann cells along the direction of the grooves was measured at 24 h after seeding the cells. When the width/spacing of the grooves was fixed at 10/10 microm, the mean percentage of aligned cells was 12% for grooves of 0.5 microm depth, 15% for those of 1 microm depth, and 26% for those of 1.5 microm depth (P < 0.05). When the depth of grooves was fixed at 1.5 microm, the mean percentage of aligned cells increased from 26% for width/spacing 10/10 microm, to 33% for 10/20 microm or 20/10 microm, and up to 41% for 20/20 microm (P < 0.05). On the surface with grooves of width/spacing/depth = 20/20/1.5 microm and modified by laminin, the alignment at 24 h approached 60%, versus 51% for collagen-coated surface and 41% for uncoated surface (P < 0.05). At 48 h after seeding, about 66% of the cells were aligned on the above laminin-modified surface. The groove depth influenced orientation of Schwann cells significantly. The cell alignment on 20/20/3 microm microgrooved poly(D,L-lactide-co-glycolide) 90:10 (PLGA) surfaces transferred from silicon reached 72% at 48 h and 92% at 72 h (P < 0.05). Coating this surface with laminin enhanced cell alignment only in short term (67% vs. 62% at 24 h, P < 0.05). The cell alignment guided by surface microgrooves was time dependent.     

ProBDNF inhibits collective migration and chemotaxis of rat Schwann cells

Schwann cell migration, including collective migration and chemotaxis, is essential for the formation of coordinate interactions between Schwann cells and axons during peripheral nerve development and regeneration. Moreover, limited migration of Schwann cells imposed a serious obstacle on Schwann cell-astrocytes intermingling and spinal cord repair after Schwann cell transplantation into injured spinal cords. Recent studies have shown that mature brain-derived neurotrophic factor, a member of the neurotrophin family, inhibits Schwann cell migration. The precursor form of brain-derived neurotrophic factor, proBDNF, was expressed in the developing or degenerating peripheral nerves and the injured spinal cords. Since “the yin and yang of neurotrophin action” has been established as a common sense, proBDNF would be expected to promote Schwann cell migration. However, we found, in the present study, that exogenous proBDNF also inhibited in vitro collective migration and chemotaxis of RSC 96 cells, a spontaneously immortalized rat Schwann cell line. Moreover, proBDNF suppressed adhesion and spreading of those cells. At molecular level, proBDNF inhibits F-actin polymerization and focal adhesion dynamics in cultured RSC 96 cells. Therefore, our results suggested a special case against the classical opinion of “the yin and yang of neurotrophin action” and implied that proBDNF might modulate peripheral nerve development or regeneration and spinal cord repair through perturbing native or transplanted Schwann cell migration.     

Ursolic acid inhibits Th17 cell differentiation via STAT3/RORγt pathway and suppresses Schwann cell-mediated Th17 cell migration by reducing CXCL9/10 expression

Th17 cells represent important immune cells. Ursolic acid (UA) can regulate immune cell activities. This study was aimed to explore the effects of UA on Th17 cell differentiation and Schwann cell(SCs)-mediated migration and the potential mechanism. Naïve CD4⁺ T cells were isolated from rat peripheral blood, induced for Th17 cell differentiation, and treated with UA. The proportion of Th17 cells was detected by flow cytometry assay. SCs were co-cultured with Th17 cells. Th17 cell migration was detected by Transwell assay. The molecule expression was determined by Western blot and qRT-PCR. UA inhibited the Th17 cell differentiation and suppressed the STAT3/RORγt pathway. STAT3 overexpression up-regulated p-STAT3 and RORγt expression and promoted Th17 cell differentiation under the UA treatment. In LPS- and IFN-γ-stimulated-SCs, UA suppressed the expression of chemokines CXCL9/10, but had no significant effect of CCL20 expression. The expression CXCL9/10 receptor CXCR3 was higher in Th17 cells than that in Treg cells, while the expression CCL20 receptor CCR6 was lower in Th17 cells than that in Treg cells. UA, anti-CXCR3, and anti-CCR6 treatment inhibited SCs-mediated Th17 cell migration, and anti-CXCR3 exerted stronger inhibitory effect than Anti-CCR6. UA inhibited Th17 cell differentiation through STAT3/RORγt pathway and suppressed Th17 cell migration through down-regulating CXCL9/10 expression in SCs.     

Glial cell line-derived neurotrophic factor-induced signaling in Schwann cells

Glial cell line-derived neurotrophic factor (GDNF), a known survival factor for neurons, has recently been shown to stimulate the migration of Schwann cells (SCs) and to enhance myelination. GDNF exerts its biological effects by activating the Ret tyrosine kinase in the presence of glycosylphosphatidylinositol-linked receptor, GDNF family receptor (GFR) alpha1. In Ret-negative cells, the alternative transmembrane coreceptor is the 140-kDa isoform of neural cell adhesion molecule (NCAM) associated with a non-receptor tyrosine kinase Fyn. We confirmed that GDNF, GFRalpha1 and NCAM are expressed in neonatal rat SCs. We found that GDNF induces an increase in the partitioning of NCAM and heparan sulfate proteoglycan agrin into lipid rafts and that heparinase inhibits GDNF-signaling in SCs. In addition to activation of extracellular signal-regulated kinases, and phosphorylation of cAMP response element binding protein, we found that cAMP-dependent protein kinase A and protein kinase C are involved in GDNF-mediated signaling in SCs. Although GDNF did not promote the differentiation of purified SCs into the myelinating phenotype, it enhanced myelination in neuron-SC cocultures. We conclude that GDNF utilizes NCAM signaling pathways to regulate SC function prior to myelination and at early stages of myelin formation.     

Brain-derived neurotrophic factor (BDNF) induces polarized signaling of small GTPase (Rac1) protein at the onset of Schwann cell myelination through partitioning-defective 3 (Par3) protein

Brain-derived neurotrophic factor (BDNF) was shown to play a role in Schwann cell myelination by recruiting Par3 to the axon-glial interface, but the underlying mechanism has remained unclear. Here we report that Par3 regulates Rac1 activation by BDNF but not by NRG1-Type III in Schwann cells, although both ligands activate Rac1 in vivo. During development, active Rac1 signaling is localized to the axon-glial interface in Schwann cells by a Par3-dependent polarization mechanism. Knockdown of p75 and Par3 individually inhibits Rac1 activation, whereas constitutive activation of Rac1 disturbs the polarized activation of Rac1 in vivo. Polarized Rac1 activation is necessary for myelination as Par3 knockdown attenuates myelination in mouse sciatic nerves as well as in zebrafish. Specifically, Par3 knockdown in zebrafish disrupts proper alignment between the axon and Schwann cells without perturbing Schwann cell migration, suggesting that localized Rac1 activation at the axon-glial interface helps identify the initial wrapping sites. We therefore conclude that polarization of Rac1 activation is critical for myelination.     

Nrg1/ErbB signaling networks in Schwann cell development and myelination

Neuregulin-1 (Nrg1) provides a key axonal signal that regulates Schwann cell proliferation, migration and myelination through binding to ErbB2/3 receptors. The analysis of a number of genetic models has unmasked fundamental mechanisms underlying the specificity of the Nrg1/ErbB signaling axis. Differential expression of Nrg1 isoforms, Nrg1 processing, and ErbB receptor localization and trafficking represent important regulatory themes in the control of Nrg1/ErbB function. Nrg1 binding to ErbB2/3 receptors results in the activation of intracellular signal transduction pathways that initiate changes in Schwann cell behavior. Here, we review data that has defined the role of key Nrg1/ErbB signaling components like Shp2, ERK1/2, FAK, Rac1/Cdc42 and calcineurin in development of the Schwann cell lineage in vivo. Many of these regulators receive converging signals from other cues that are provided by Notch, integrin or G-protein coupled receptors. Signaling by multiple extracellular factors may act as key modifiers and allow Schwann cells at different developmental stages to respond in distinct manners to the Nrg1/ErbB signal.     

Characterization of glial cell line-derived neurotrophic factor family receptor alpha-1 in peripheral nerve Schwann cells

Glial cell line-derived neurotrophic factor (GDNF) family receptor alpha-1 (GFRalpha-1) is a receptor component of GDNF that associates with and activates the tyrosine kinase receptor Ret. To further understand GDNF and its receptor system in the PNS, we first characterized the expression of GFRalpha-1 in bovine peripheral nerve in vivo. GFRalpha-1 immunoreactivity was localized adjacent to the outermost layer of myelin sheath, as well as in the endoneurium and axoplasm. In a fractionation study, GFRalpha-1 was recovered mostly in the soluble fraction, although a small amount was recovered in the membrane fraction. A substantial amount of GFRalpha-1 in the membrane fraction was extractable by detergent and alkaline conditions. To further clarify the expression of GFRalpha-1 in Schwann cells, we examined cultured rat Schwann cells and the Schwannoma cell line RT4. Schwann cells expressed GFRalpha-1 in both the soluble/cytosolic and membrane fractions, and the membrane form of GFRalpha-1 was expressed at the outer surface of the Schwann cell plasma membrane. We also confirmed the secretion of the soluble form of GFRalpha-1 from Schwannoma cells in a metabolic labeling experiment. These data contribute to our knowledge of the production, expression and functions of GFRalpha-1 in the PNS.     

Schwann cells promote endothelial cell migration

Directed cell migration is a crucial orchestrated process in embryonic development, wound healing, and immune response. The underlying substrate can provide physical and/or chemical cues that promote directed cell migration. Here, using electrospinning we developed substrates of aligned poly(lactic-co-glycolic acid) nanofibres to study the influence of glial cells on endothelial cells (ECs) in a 3-dimensional (3D) co-culture model. ECs build blood vessels and regulate their plasticity in coordination with neurons. Likewise, neurons construct nerves and regulate their circuits in coordination with ECs. In our model, the neuro-vascular cross-talk was assessed using a direct co-culture model of human umbilical vein endothelial cells (HUVECs) and rat Schwann cells (rSCs). The effect of rSCs on ECs behavior was demonstrated by earlier and higher velocity values and genetic expression profiles different of those of HUVECs when seeded alone. We observed 2 different gene expression trends in the co-culture models: (i) a later gene expression of angiogenic factors, such as interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF), and (ii) an higher gene expression of genes involved in actin filaments rearrangement, such as focal adhesion kinase (FAK), Mitogen-activated protein kinase-activated protein kinase 13 (MAPKAPK13), Vinculin (VCL), and Profilin (PROF). These results suggested that the higher ECs migration is mainly due to proteins involved in the actin filaments rearrangement and in the directed cell migration rather than the effect of angiogenic factors. This co-culture model provides an approach to enlighten the neurovascular interactions, with particular focus on endothelial cell migration.