Differential expression of HNK-1 and p75(NTR) in adult canine Schwann cells and olfactory ensheathing cells in situ but not in vitro

Olfactory ensheathing cells (OECs) are promising candidates for autologous cell transplantation therapies of nervous system injury and disease. Large animal models are relevant for transferring experimental data into clinical practice. In vivo studies have suggested that adult canine OECs may display similar regenerating capacities as their rodent counterpart. However, data on their molecular phenotype required for generating pure cell preparations are still scarce. In the present study, we comparatively analyzed expression of the carbohydrate HNK-1 epitope and the neurotrophin receptor p75(NTR) in adult canine Schwann cells and olfactory ensheathing cells in situ and in vitro. Myelinating and nonmyelinating Schwann cells in situ exclusively expressed HNK-1 and p75(NTR), respectively, whereas OECs were negative for both markers. In vitro, OECs and Schwann cells shared cell surface expression of p75(NTR) but not of HNK-1, which could be detected transiently in intracellular vesicles. This suggests that Schwann cells and OECs in vitro phagozytose HNK-1+ cellular debris. The cultivation-induced downregulation of HNK-1 expression in Schwann cells and upregulation of p75(NTR) in OECs argues for the possibility that axonal signals control the expression of both markers in situ. Whereas HNK-1 expression in Schwann cells is most likely controlled by signals inducing myelination, e.g., neuregulin, the mechanisms that may suppress p75(NTR) expression in OECs in situ remain to be elucidated. Interestingly, HNK-1 expression in the adult dog was found in both sensory and motor nerve myelinating Schwann cells. This is reminiscent of humans and differs from rodents; it also underscores the importance of large animal models for translational research.     

GABAB receptors in Schwann cells influence proliferation and myelin protein expression

The location and the role of gamma-aminobutyric acid type B (GABA(B)) receptors in the central nervous system have recently received considerable attention, whilst relatively little is known regarding the peripheral nervous system. In this regard, here we demonstrate for the first time that GABA(B) receptor isoforms [i.e. GABA(B(1)) and GABA(B(2))] are specifically localized in the rat Schwann cell population of the sciatic nerve. Using the selective GABA(B) agonist [i.e. (-)-baclofen] and the antagonists (i.e. CGP 62349, CGP 56999 A, CGP 55845 A), such receptors are shown to be functionally active and negatively coupled to the adenylate cyclase system. Furthermore, exposure of cultured Schwann cells to (-)-baclofen inhibits their proliferation and reduces the synthesis of specific myelin proteins (i.e. glycoprotein Po, peripheral myelin protein 22, myelin-associated glycoprotein, connexin 32), providing evidence for a physiological role of GABA(B) receptors in the glial cells of the peripheral nervous system.     

Schwann cells depleted of galactocerebroside express myelin-associated glycoprotein and initiate but do not continue the process of myelination.

Two peripheral myelin components, galactocerebroside (GalC) and myelin-associated glycoprotein (MAG), are known to be expressed early in Schwann cell differentiation, prior to the formation of definitive myelin segments containing compacted membrane. To discern the relative roles of these myelin components, cultures of Schwann cells and dorsal root ganglion neurons were treated with antigalactocerebroside mAbs in order to remove GalC from the Schwann cell surface (Ranscht et al., 1987). In the continuous presence of anti-GalC antibodies and in a medium containing serum plus ascorbic acid, Schwann cells assemble a basal lamina and progress to the one:one stage of Schwann cell:axon interaction but do not differentiate further. Immunostaining with anti-MAG antibodies revealed that GalC-depleted Schwann cells expressed high levels of MAG. Double staining with anti-MAG and anti-P0 antibodies showed that there was essentially no P0 immunoreactivity in the same cells. In those Schwann cells that had attained a one:one association with large-diameter axons, the inner-axon-related cytoplasmic process had passed under the outer mesaxon but had not completed a full turn around the axon. The expression of MAG on the single cytoplasmic process apposed to the axon in Schwann cells depleted of GalC further implicates MAG in the initial envelopment of the axon during myelination.     

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

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

Transplanted Schwann cells, not olfactory ensheathing cells, myelinate optic nerve fibres

In a previous study we found that olfactory ensheathing cells transplanted into complete retrobulbar transections of the rat optic nerve mediated regeneration of severed retinal ganglion cell axons through the graft region. Although the regenerating axons were ensheathed by the transplanted cells, none of the regenerating axons became myelinated by either central or peripheral type myelin. In the present study we used the same operative procedure but transplanted Schwann cells instead of olfactory ensheathing cells. As with the olfactory ensheathing cell transplants the Schwann cells transplants also induced regeneration of the severed retinal ganglion cell axons into the graft region. In contrast to the situation with the olfactory ensheathing cell transplants, however, a considerable number of the regenerating axons became myelinated by peripheral type myelin produced by the transplanted Schwann cells. This observation identifies a further distinction between these two cell types which are phenotypically similar in many ways, but which have been shown to have major functional differences with regard to regeneration in spinal cord lesions.     

Elevated intracellular levels of cAMP induce olfactory ensheathing cells to express GAL-C and GFAP but not MBP

The primary olfactory pathway contains non-myelinating glial cells, called ensheathing cells, that exhibit a variety of phenotypes depending on their immediate environment. In vivo, these cells normally possess a mixture of astrocyte- and Schwann cell-specific phenotypic features. When co-cultured with dorsal root ganglion neurons, their phenotype can become more like that of a myelinating Schwann cell. The objective of this study was to determine whether ensheathing cells would express a myelinating phenotype in culture in the absence of neurons but in the presence of cAMP analogues that are known to induce the expression of myelin associated molecules in Schwann cell cultures. The ensheathing cell cultures were initiated using the nerve fiber layers of Theiler stage 23 rat olfactory bulb primordia and were fed for 1 day to 3 weeks with serum containing (1% or 10% FBS) or serum-free media to which was added different concentrations of dBcAMP (0.1 to 1 mM) or forskolin (10 M). These cultures were double-labelled with a rabbit polyclonal antibody to S100 in combination with mouse anti-GAL-C (01 and BRD1 hybridomas) or anti-MBP monoclonal antibodies. The remaining cultures were double-labeled with a rabbit polyclonal antibody to GFAP in combination with the BRD1 antibody. Treatment with dBcAMP or forskolin failed to induce ensheathing cells to express MBP regardless of the concentration. On the other hand, the treatment induced approximately one tenth of the cells to express GAL-C, and virtually all of the cells to express GFAP. These results indicate that although ensheathing cells can synthesize myelin associated molecules, the cAMP second messenger system appears to play a lesser role in controlling the expression of a myelinating phenotype in ensheathing cells than it does in Schwann cells. © 1995 Wiley-Liss, Inc.     

Olfactory ensheathing cells express smooth muscle alpha-actin in vitro and in vivo

One strategy for spinal cord repair after injury that has moved quickly from the research laboratory to the clinic is the implantation of olfactory ensheathing cells (OECs). These unique glial cells of the olfactory system have been associated with axonal remyelination and regeneration after grafting into spinalized animals. Despite these promising observations, there remains a lack of direct empirical evidence of the exact fate of OECs after intraspinal implantation, in large part because of a surprising paucity of defined biomarkers that unequivocally distinguish these cells from phenotypically similar Schwann cells. Here we provide direct neurochemical proof that OECs, both in vitro and in vivo, express smooth muscle alpha-actin. That OECs synthesize this contractile protein (and a variety of actin-binding proteins including caldesmon) provides compelling evidence that these cells are, in fact, quite different from Schwann cells. The identification of several smooth muscle-related proteins in OECs points to a new appreciation of the structural and functional features of this population of olfactory glia. These biomarkers can now be used to elucidate the fate of OECs after intraspinal implantation, in particular assessing whether smooth muscle alpha-actin-expressing OECs are capable of facilitating axon remyelination and regeneration.     

Media that support the growth and differentiation of oligodendrocytes do not induce olfactory ensheathing cells to express a myelinating phenotype

The glial cells that ensheath olfactory axons are referred to as ensheathing cells. In vivo, these non-myelinating glial cells express a mixture of astrocyte-specific and Schwann cell-specific phenotypic features with the former cellular phenotype predominating, but in vitro can assemble a myelin sheath when co-cultured with dorsal root ganglion neurons. Thus, certain in vitro conditions induce ensheathing cells to express a phenotype more like that of a myelinating Schwann cell. The present study addresses whether ensheathing cells will express a myelinating phenotype in neuron-free cultures when fed for 1 to 5 weeks with media shown to promote the growth and differentiation of oligodendrocyte progenitor cells. The ensheathing cell cultures were initiated using the nerve fiber layers (NFL) of rat olfactory bulb primordia. Oligodendrocyte cultures were established from newborn rat neopallium and from the tissue that remained after removing the NFL from the developing olfactory bulb (i.e., the OB-NFL). The cultures were double-labelled with rabbit polyclonal antibodies to S100 or glial fibrillary acidic protein in combination. With the mouse monoclonal antibodies 04, BRD1 [anti-galactocerebroside (anti-GAL-C)], and anti-myelin basic protein (MBP). In some experiments the ensheathing cells were labelled with PKH26 prior to being co-cultured with oligodendrocytes of the OB-NFL. None of the media induced ensheathing cells to express either GAL-C or MBP. However, when 0.5 mM dibutyrylcyclic-AMP (dBcAMP) was added to the medium, ensheathing cells became GAL-C + ve, but remained MBP-ve. The molecular mechanisms that regulate the expression of a myelinating phenotype by ensheathing cells appear to be different from those that operate in oligodendrocytes. © 1994 Wiley-Liss, Inc.     

Macrophages but not Schwann cells express Ia antigen in experimental autoimmune neuritis

This study was designed to identify which cells express major histocompatibility complex class II (Ia) antigen in experimental autoimmune neuritis and may therefore be antigen presenters. Serial 1-micron-thick cryosections of ventral roots of animals with experimental autoimmune neuritis were labeled with Ox6 antibody against rat Ia, the ED1 antibody to identify monocytes/macrophages and an antiserum against S100, a marker for Schwann cells. Ia-positive cells were predominantly present before overt clinical signs and demyelination (day 12). At later stages when many axons were demyelinated, their number was markedly reduced. Few Ia-positive cells that had extending long processes, which over some distance were in immediate contact with several myelin sheaths, were scattered in normal-appearing nerve roots at these later time points. Most of the Ia-positive cells could be identified as ED1-positive lean monocytes/macrophages, but in contrast most phagocytic macrophages in advanced stages of myelin degradation no longer expressed Ia. Ia-positive structures were invariably negative for S100 at early and late stages of experimental autoimmune neuritis, indicating that Schwann cells did not express identifiable Ia antigen. These findings contrast with reports of expression of major histocompatibility complex class II antigens by Schwann cells in human neuropathies. Furthermore they do not support the notion that aberrant Ia expression by Schwann cells plays a major pathogenic role in experimental autoimmune disease of the peripheral nervous system.     

Myelin-specific proteolipid protein is expressed in myelinating Schwann cells but is not incorporated into myelin sheaths

Contrary to widely held beliefs, the gene encoding proteolipid protein (PLP), the major structural protein of central nervous system myelin, is expressed in Schwann cells and their tumors. Proteolipid mRNA was identified in human acoustic neuromas and in rat and rabbit sciatic nerves using a human PLP cDNA as a probe. Proteolipid protein itself was shown to be present in human and rat sciatic nerve Schwann cells by immunofluorescence microscopy and by Western blot analysis using antisera raised to a synthetic PLP polypeptide. Although easily detected in the Schwann cell body, PLP was not detected in the peripheral myelin itself, suggesting that the PLP is preferentially excluded from this portion of the Schwann cell membrane.     

Calponin is expressed by fibroblasts and meningeal cells but not olfactory ensheathing cells in the adult peripheral olfactory system

Olfactory ensheathing cells (OECs), the principal glial cells of the peripheral olfactory system, have many phenotypic similarities with Schwann cells of the peripheral nervous system. This makes reliably distinguishing these two cells types difficult, especially following transplantation into areas of injury in the central nervous system. In an attempt to identify markers by which these two cells types can be distinguished, a recent proteomic analysis of fetal OECs and adult Schwann cells identified the actin-binding protein calponin as a potential marker expressed by OECs but not Schwann cells. Since many studies designed with the translational goal of autologous transplantation in mind have used adult OECs, this study examined the expression of calponin by adult OECs, both in vivo within the peripheral olfactory system and in vitro. Calponin colocalized with strongly fibronectin positive fibroblasts in the olfactory mucosa (OM) and meningeal cells in the olfactory bulb (OB) but not with S100beta or neuropeptide-Y positive OECs. In tissue culture, calponin was strongly expressed by fibronectin-expressing fibroblasts from OM, sciatic nerve and skin and by meningeal cells from the OB, but not by p75(NTR)- and S100beta-expressing OECs. These data, supported by Western blotting, indicate that calponin can not be used to distinguish adult OECs and Schwann cells.     

Axon-glia communication evokes calcium signaling in olfactory ensheathing cells of the developing olfactory bulb

Olfactory ensheathing cells (OECs) accompany receptor axons in the olfactory nerve and promote axonal growth into the central nervous system. The mechanisms underlying the communication between axons and OECs, however, have not been studied in detail yet. We investigated the effect of activity-dependent neuronal transmitter release on Ca(2+) signaling of OECs in acute mouse olfactory bulb slices using confocal Ca(2+) imaging. TTX-sensitive axonal activity upon electrical nerve stimulation triggers a rise in cytosolic Ca(2+) in OECs, which can be mimicked by application of DHPG, an agonist of metabotropic glutamate receptors (mGluRs). Both stimulation- and DHPG-induced Ca(2+) transients in OECs were abolished by depletion of intracellular Ca(2+) stores with cyclopiazonic acid (CPA). The mGluR(1)-specific antagonist CPCCOEt completely inhibited DHPG-evoked Ca(2+) transients, but reduced stimulation-induced Ca(2+) transients only partly, suggesting the involvement of another neurotransmitter. Application of ATP evoked CPA-sensitive Ca(2+) transients in OECs, which were inhibited by the P2Y(1)-specific antagonist MRS2179. Co-application of CPCCOEt and MRS2179 almost completely blocked the stimulation-induced Ca(2+) transients, indicating that they were mediated by mGluR(1) and P2Y(1) receptors. Our results show that OECs are able to respond to olfactory nerve activity with an increase in cytosolic Ca(2+) due to glutamate and ATP release.     

Response of olfactory Schwann cells to intranasal zinc sulfate irrigation

The response of olfactory Schwann cells was assessed at 2, 4, and 7 days following intranasal zinc sulfate irrigation in 1-month-old mice. Ultrastructural and immunohistochemical observations showed dramatic differences between experimental and control mice which had been washed with saline intranasally. Two days after zinc sulfate treatment, many olfactory nerve bundles contained patchy areas of axonal degeneration, while the cell bodies of the olfactory Schwann cells appeared to have increased in electron density and to have shifted peripherally. Some of the cell bodies protruded from the surface of the axon fascicle, suggesting that the olfactory Schwann cells were in the initial process of migrating away. On the fourth day when most of the olfactory axons had degenerated, some olfactory Schwann cells were aligned immediately beneath the basal lamina of the olfactory epithelium. These cells were immunopositive for the S-100 protein and possessed an expanded perinuclear space. Many olfactory Schwann cells were present in the region beneath the cribriform plate, while some appeared to have passed through the gaps between the bony plates to reach the olfactory bulb. Hence, the results showed that many olfactory Schwann cells migrated towards the olfactory bulb following loss of axonal contact. Furthermore, on the seventh day following zinc sulfate treatment, some olfactory Schwann cells in the vicinity of the olfactory bulb appeared phagocytic, as indicated by their extension of processes around fragments of cell debris and the presence of lysosome-like organelles in the perikaryon. The control mice which had been intranasally irrigated with saline did not demonstrate massive olfactory axonal degeneration, and the morphology of the nasal cavity region was similar to that of normal mice. © 1995 Wiley-Liss, Inc.     

Olfactory ensheathing cells promote neurite extension from embryonic olfactory receptor cells in vitro

The role of ensheathing cells, a macroglial cell type with a unique presence in the olfactory system, in the outgrowth of olfactory receptor cell neurites was explored in vitro. Glial cell cultures harvested from both the olfactory bulb nerve layer and the hippocampus were established and immunocytochemically characterized. The expression of the p75 low‐affinity nerve growth factor receptor by ensheathing cells was used to distinguish them from other macroglial subpopulations. Results indicated that ensheathing cell cultures were approximately 80% pure. Olfactory receptor cells were cocultured with ensheathing or hippocampal glial cells or were seeded on laminin or poly‐l‐ lysine as controls. Olfactory receptor cells extended the longest primary neurites when cocultured with ensheathing cells. Neurite extension on hippocampal glia and laminin was less extensive than that observed on ensheathing cells but higher than that on poly‐l‐ lysine. The neurite outgrowth‐promoting effect of ensheathing cells was, at least in part, mediated by diffusible factors, because olfactory receptor cell neurite extension could also be facilitated when receptor cells were cultured in ensheathing cell‐conditioned media. In contrast, cortical neurons extended neurites of equivalent lengths on ensheathing and hippocampal glia. The results suggest that ensheathing cells may release factors that support the continuous outgrowth of olfactory receptor cell axons and, therefore, the capacity of this pathway to recover from injury. GLIA 25:99–110, 1999. © 1999 Wiley‐Liss, Inc.     

Morphological plasticity of olfactory ensheathing cells is regulated by cAMP and endothelin-1

Olfactory ensheathing cells (ECs) are a promising tool for the repair of injury in the adult central nervous system. However, important aspects of the cell biology of ECs remain unclear, such as whether ECs exist as a single population or as two subpopulations with Schwann cell-like and astrocyte-like characteristics. The morphologies of these subpopulations are used as defining characteristics, yet ECs are known to be morphologically plastic. To elucidate this apparent inconsistency, we investigated the morphological plasticity of ECs in culture. We defined purified ECs as immunopositive for both p75 neurotrophin receptor and glial fibrillary acidic protein. In MEM (D)-valine modification + 10% dialyzed fetal calf serum, 87%-90% of ECs displayed a flat morphology. In three different serum-free media (N2 medium, neurobasal medium + B27 supplement, and DMEM/F-12 medium + G5 supplement), 78%-84% of ECs displayed process-bearing morphology. Ensheathing cells switched reversibly between these morphologies within a day of the serum conditions being changed. Exposure to 1 nM endothelin-1 in serum-free medium prevented the switch from flat to process-bearing morphology, while 1 mM dibutyryl cAMP accelerated this change. The effects of both agents were completely reversible and similar to that reported for astrocytes. Both flat and process-bearing ECs were immunopositive for brain-derived neurotrophic factor, nerve growth factor, neurotrophin-4, and TrkB but not TrkA. Together, these results suggest that ECs exist as a single morphologically plastic population.     

Voltage‐dependent K+ currents contribute to heterogeneity of olfactory ensheathing cells

The olfactory nerve is permissive for axon growth throughout life. This has been attributed in part to the olfactory ensheathing glial cells that encompass the olfactory sensory neuron fascicles. Olfactory ensheathing cells (OECs) also promote axon growth in vitro and when transplanted in vivo to sites of injury. The mechanisms involved remain largely unidentified owing in part to the limited knowledge of the physiological properties of ensheathing cells. Glial cells rely for many functions on the properties of the potassium channels expressed; however, those expressed in ensheathing cells are unknown. Here we show that OECs express voltage‐dependent potassium currents compatible with inward rectifier (K_ir) and delayed rectifier (K_DR) channels. Together with gap junction coupling, these contribute to the heterogeneity of membrane properties observed in OECs. The relevance of K⁺ currents expressed by ensheathing cells is discussed in relation to plasticity of the olfactory nerve. GLIA 2015;63:1646–1659     

Downregulation of steroidogenic acute regulatory protein (StAR) gene expression by cyclic AMP in cultured Schwann cells

Steroidogenic acute regulatory protein (StAR) plays a key role in the availability of cholesterol to the inner mitochondrial membrane, where the first step of steroidogenesis, its conversion to pregnenolone, takes place. Here, we demonstrate for the first time that the StAR gene is also expressed in the rat sciatic nerve and in cultured Schwann cells. The addition to the culture medium of the cAMP-elevating agent forskolin or of the cAMP analogue 8Br-cAMP produced a time-course extinction of StAR gene expression. An inverse relationship was demonstrated between StAR gene expression and the intracellular cAMP content. Accordingly, pharmacological inhibition of the activities of Schwann cell adenylyl cyclase or of phosphodiesterase IV resulted in modifications of StAR gene expression. Since StAR gene expression is stimulated by cAMP in classical steroidogenic cells, our work is the first demonstration of a negative regulation of StAR gene by cAMP.