Nonmyelin-forming Schwann Cells Research Articles

An update on Schwann cell biology — Immunomodulation, neural regulation and other surprises

Schwann cells are primarily discussed in the context of their ability to form myelin. However there are many subtypes of these neural crest derived cells including satellite cells of the dorsal root ganglia and autonomic ganglia, the perisynaptic Schwann cells of the neuromuscular junction and the non-myelin forming Schwann cells which ensheathe the unmyelinated fibres of the peripheral nervous system which are about 80% of peripheral nerves. This review discusses the many functions of these Schwann cell subsets including their seminal role in axonal ensheathment, perineuronal organisation, maintenance of normal neural function, synapse formation, response to damage and repair and an increasingly recognised active role in pain syndromes.

Both Schwann cell and axonal defects cause motor peripheral neuropathy in Ebf2−/− mice

Charcot–Marie–Tooth neuropathies are frequent hereditary disorders of the nervous system and most cases remain without a molecular definition. Mutations in transcription factors have been previously associated to various types of this disease. Mice carrying a null mutation in Ebf2 transcription factor present peripheral nerve abnormalities. To get insight into Ebf2 function in peripheral nervous system, here we characterize the peripheral neuropathy affecting these mice. We first show that Ebf2 is largely expressed in peripheral nerve throughout postnatal development, its expression being not only restricted to non-myelin forming Schwann cells, but also involving myelin forming Schwann cells and the perineurium. As a consequence, the onset of myelination is delayed and Schwann cell differentiation markers are downregulated in Ebf2−/− mice. Later in development, myelin pathology appears less severe and characterized by isolated clusters of hypomyelinated fibers. However, we find defects in the nerve architecture, such as abnormalities of the nodal region and shorter internodal length. Furthermore, we demonstrate a significant decrease in axonal calibre, with a lack of large calibre axons, and a severe impairment of motor nerve conduction velocity and amplitude, whereas the sensory nerve parameters are less affected. Interestingly, a clinical case with peripheral motor neuropathy and clinical features similar to Ebf2−/− mice phenotype was associated with a deletion encompassing EBF2 human genomic locus. These findings demonstrate that Ebf2 is a new molecule implicated in peripheral nerve development and a potential candidate gene for peripheral nerve disorders.

Biology and pathology of nonmyelinating Schwann cells

The CNS contains relatively few unmyelinated nerve fibers, and thus benefits from the advantages that are conferred by myelination, including faster conduction velocities, lower energy consumption for impulse transmission, and greater stability of point-to-point connectivity. In the PNS many fibers or regions of fibers the Schwann do not form myelin. Examples include C fibers nociceptors, postganglionic sympathetic fibers, and the Schwann cells associated with motor nerve terminals at neuromuscular junctions. These examples retain a degree of plasticity and a capacity to sprout collaterally that is unusual in myelinated fibers. Nonmyelin-forming Schwann cells, including those associated with uninjured fibers, have the capacity to act as the "first responders" to injury or disease in their neighborhoods.

Changes of Schwann cell antigenic profile after peripheral nerve injury.

Axons and Schwann cells (SC) in the peripheral nerve (PN) are in permanent interaction. Both myelin-forming SC (MSC) and nonmyelin-forming SC (NMSC) have a defined antigenic phenotype. Four weeks after PN transection, the proliferating SC in the distal stump lose some of their antigens, they start to display antigenic profile of NMSC and, in addition, to reexpress nerve growth factor receptor (NGF-rec) and growth associated protein (GAP-43), two antigens of their precursor cells. It can be assumed that migrating SC may differ in their antigenic profile in regard to resident proliferated SC in denervated distal nerve stump. We found that many SC specific antigens (S-100 protein, glial fibrilary acidic protein-GFAP, NGF-rec, GAP-43) are down-regulated in migrating SC as compared to SC proliferating in situ in the absence of axons.

Expression of the 4C5 antigen during development and after injury of the rat sciatic nerve

We have previously reported the production of monoclonal antibody 4C5 that recognizes a cell surface neuron specific antigen, the 4C5 antigen (Thomaidou and Patsavoudi: Neuroscience 53:813–827, 1993). Antibody perturbation experiments have suggested that the 4C5 antigen is involved in granule cell migration processes in vitro (Thomaidou et al: J Neurochem 64:1937–1944, 1995). In the present study, we have used monoclonal antibody 4C5 to investigate the cellular distribution and the expression of the corresponding antigen during development, and after injury of the rat sciatic nerve. Immonocytochemistry revealed presence of the protein both in myelin and non-myelin forming Schwann cells. Western blot analysis showed that monoclonal antibody 4C5 recognizes the same 94 kdalton polypeptide in the sciatic nerve as in the brain. Immunocytochemical analysis revealed an age-dependent decline of the 4C5 antigen expression. Moreover, following adult sciatic nerve crush, 4C5 immunoreactivity becomes intense in Schwann cells of the distal segment, as early as 4 days after injury and remains at relatively high levels up to 2 weeks after the crush. By the 3rd week, 4C5 immunoreactivity is greatly reduced, and by the 4th week it is hardly detectable. Our results concerning the cellular localization and temporal expression of the 4C5 antigen during development and after injury of the rat sciatic nerve, suggest that it may participate in Schwann cell migrations that occur during such processes. © 1996 Wiley-Liss, Inc.

Expression of the 4C5 antigen during development and after injury of the rat sciatic nerve.

We have previously reported the production of monoclonal antibody 4C5 that recognizes a cell surface neuron specific antigen, the 4C5 antigen (Thomaidou and Patsavoudi: Neuroscience 53:813-827, 1993). Antibody perturbation experiments have suggested that the 4C5 antigen is involved in granule cell migration processes in vitro (Thomaidou et al: J Neurochem 64:1937-1944, 1995). In the present study, we have used monoclonal antibody 4C5 to investigate the cellular distribution and the expression of the corresponding antigen during development, and after injury of the rat sciatic nerve. Immunocytochemistry revealed presence of the protein both in myelin and non-myelin forming Schwann cells. Western blot analysis showed that monoclonal antibody 4C5 recognizes the same 94 kdalton polypeptide in the sciatic nerve as in the brain. Immunocytochemical analysis revealed an age-dependent decline of the 4C5 antigen expression. Moreover, following adult sciatic nerve crush, 4C5 immunoreactivity becomes intense in Schwann cells of the distal segment, as early as 4 days after injury and remains at relatively high levels up to 2 weeks after the crush. By the 3rd week, 4C5 immunoreactivity is greatly reduced, and by the 4th week it is hardly detectable. Our results concerning the cellular localization and temporal expression of the 4C5 antigen during development and after injury of the rat sciatic nerve, suggest that it may participate in Schwann cell migrations that occur during such processes.

Mice lacking glial fibrillary acidic protein display astrocytes devoid of intermediate filaments but develop and reproduce normally.

Glial fibrillary acidic protein (GFAP) is the main component of the intermediate filaments in cells of astroglial lineage, including astrocytes in the CNS, nonmyelin forming Schwann cells and enteric glia. To address the function of GFAP in vivo, we have disrupted the GFAP gene in mice via targeted mutation in embryonic stem cells. Mice lacking GFAP developed normally, reached adulthood and reproduced. We did not find any abnormalities in the histological architecture of the CNS, in their behavior, motility, memory, blood-brain barrier function, myenteric plexi histology or intestinal peristaltic movement. Comparisons between GFAP and S-100 immunohistochemical staining patterns in the hippocampus of wild-type and mutant mice suggested a normal abundance of astrocytes in GFAP-negative mice, however, in contrast to wild-types, GFAP-negative astrocytes of the hippocampus and in the white matter of the spinal cord were completely lacking intermediate filaments. This shows that the loss of GFAP intermediate filaments is not compensated for by the up-regulation of other intermediate filament proteins, such as vimentin. The GFAP-negative mice displayed post-traumatic reactive gliosis, which suggests that GFAP up-regulation, a hallmark of reactive gliosis, is not an obligatory requirement for this process.

Immunocytochemical localization of a growth-associated protein (GAP-43) in rat adrenal gland.

We have localized at light and electron-microscopic level the growth-associated protein GAP-43 in adrenal gland using single and double labelling immunocytochemistry. Clusters of GAP-43-immunofluorescent chromaffin cells and many immunofluorescent fibres were observed in the medulla. GAP-43-immunoreactive fibres also formed a plexus under the capsule, crossed the cortex and ramified in the zona reticulata. Double labelled sections showed the coexpression of GAP-43 with a subpopulation of tyrosine hydroxylase- and of dopamine-beta-hydroxylase-immunoreactive chromaffin cells. Dual colour immunofluorescence for GAP-43 and calcitonin gene-related peptide (CGRP) revealed that some of the GAP-43-immunoreactive fibres also express CGRP. Pre-embedding electron microscopy showed GAP-43 immunoreactivity associated with the plasma membranes and cytoplasm of noradrenaline-producing chromaffin cells, and with processes of nonmyelin-forming Schwann cells. Immunoreactive unmyelinated axons and terminals were also observed. The immunostained terminals made symmetrical synaptic contacts with chromaffin cells. Immunoreactive unmyelinated fibres and small terminals were present in the cortex. Our results show that GAP-43 is expressed in noradrenergic chromaffin cells and in various types of nerve fibres that innervate the adrenal. Likely origins for these fibres include preganglionic sympathetic fibres which innervate chromaffin cells, postganglionic sympathetic fibres in the cortex, and CGRP containing sensory fibres.

Human peripheral nerve macrophages in normal and pathological conditions

We investigated, by immunocytochemistry and immune electron microscopy, the immunophenotype, morphology and functional properties of human peripheral nervous system (PNS) macrophages (MΦ) under normal and pathological conditions. Endoneurial MΦ disclosed an elongated, ramified morphology, with the main processes oriented along the major axis of nerve fibers; they shared several lineage-related and functional markers with monocyte/macrophages and central nervous system (CNS) microglia, including CD4, CR3, CR4 and FcRIII. In addition, basal expression of HLA-DR antigens was exclusively confined to MΦ in normal PNS. In the course of unrelated pathological conditions, resident MΦ underwent activation with transformation to hypertrophic cells or foamy phagocytes and up-regulation of the markers expressed in normal conditions; new expression of a macrophagic antigen was detected on activated MΦ. In different neuropathies, HLA-DR expression was also detected on non-myelin forming Schwann cells with ultrastructural features indicative of denervation. The present results demonstrate that the human PNS is provided with an intrinsic population of immunocompetent and potentially phagocytic MΦ, which represent the peripheral counterpart of CNS microglia.

Changes in DNA synthesis rate in the Schwann cell lineage in vivo are correlated with the precursor--Schwann cell transition and myelination.

During the development of the rat sciatic nerve extensive proliferation of glial cells occurs, and there is a very substantial rearrangement of the cytoarchitecture as axons and Schwann cells assume relationships which lead to the formation of the myelinated and unmyelinated axons characteristic of adult nerve. The maturation of Schwann cells from Schwann cell precursors and the matching of Schwann cell numbers to axons is an important part of this process. We have therefore studied the proliferation of Schwann cell precursors and Schwann cells during the development of the rat sciatic nerve from embryonic day 14 to postnatal day 28 by combining bromodeoxyuridine injections of rats with double-label immunohistochemical techniques. The results reveal that DNA synthesis occurs in both Schwann cell precursors and Schwann cells throughout early nerve development. The labelling index is already substantial at embryonic day 14, but from embryonic day 17, when essentially all the glial cells have converted from precursor to Schwann cell phenotype, it rises sharply, peaking between embryonic day 19 and 20 before declining precipitously in the early postnatal period. This rapid decline in DNA synthesis coincides with the appearance of the myelin protein P0, and in individual cells DNA synthesis is incompatible with the expression of P0 protein. Nonmyelin-forming Schwann cells, which mature later in development, continue to synthesize DNA until at least postnatal day 15, but by day 28 essentially all Schwann cells in the nerve are quiescent.

GAP-43 is expressed by nonmyelin-forming Schwann cells of the peripheral nervous system.

Recently it has been demonstrated that the growth-associated protein GAP-43 is not confined to neurons but is also expressed by certain central nervous system glial cells in tissue culture and in vivo. This study has extended these observations to the major class of glial cells in the peripheral nervous system, Schwann cells. Using immunohistochemical techniques, we show that GAP-43 immunoreactivity is present in Schwann cell precursors and in mature non-myelin-forming Schwann cells both in vitro and in vivo. This immunoreactivity is shown by Western blotting to be a membrane-associated protein that comigrates with purified central nervous system GAP-43. Furthermore, metabolic labeling experiments demonstrate definitively that Schwann cells in culture can synthesize GAP-43. Mature myelin-forming Schwann cells do not express GAP-43 but when Schwann cells are removed from axonal contact in vivo by nerve transection GAP-43 expression is upregulated in nearly all Schwann cells of the distal stump by 4 wk after denervation. In contrast, in cultured Schwann cells GAP-43 is not rapidly upregulated in cells that have been making myelin in vivo. Thus the regulation of GAP-43 appears to be complex and different from that of other proteins associated with nonmyelin-forming Schwann cells such as N-CAM, glial fibrillary acidic protein, A5E3, and nerve growth factor receptor, which are rapidly upregulated in myelin-forming cells after loss of axonal contact. These observations suggest that GAP-43 may play a more general role in the nervous system than previously supposed.

Cellular reorganisation of membrane peptidases in Wallerian degeneration of pig peripheral nerve

Immunohistochemical techniques have been used to study a group of membrane peptidases in the distal segment of the ulnar nerve of piglets 7 and 14 days after surgical section. Five peptidases were studied, all of which have a wide distribution on the surfaces of many cell types and have roles in metabolising neuropeptides. In normal pig nerves, endopeptidase-24.11 is expressed by both myelin- and nonmyelin-forming Schwann cells. Peptidyl dipeptidase A (angiotensin converting enzyme), aminopeptidase-N and dipeptidyl peptidase IV are present in the microvessels, and aminopeptidase-N is also seen in the perineurial connective tissue. Of this group of peptidases, only aminopeptidase-W is a neuronal marker in normal nerve. Macrophages were identified by two antibodies, 74-22-15 and 40D (which recognises Ia). Short-term cultures of macrophages obtained by alveolar lavage were positively stained by both antibodies and about half of the cells also expressed aminopeptidase-N and dipeptidyl peptidase IV. Staining by 40D and 74-22-15 revealed the presence of significant numbers of macrophages in normal nerve, but none of the membrane peptidases colocalized with these cells. Seven days after section of the nerve, the distal segment showed morphological changes typical of Wallerian degeneration. Endopeptidase-24.11 was no longer visible in myelin sheaths, but remained a marker for the surface of Schwann cells (defined also by staining for glial fibrillary acidic protein). The macrophage markers revealed marked changes in the morphology of these cells, often consistent with their phagocytic activity. Two peptidases, aminopeptidase-N and aminopeptidase-W, also appeared at this time to be associated with cells exhibiting the morphology of activated macrophages. This association could be confirmed in many instances by double staining with 74-22-15 and antibodies to the peptidases. Angiotensin converting enzyme retained its single location in microvessels at 7 days after section, but at 14 days a new pattern emerged as it, too, was expressed by macrophages. Dipeptidyl peptidase IV was not shown to be a macrophage marker in the degenerating nerve. Thus Wallerian degeneration leads to remarkable changes in the cellular expression of membrane peptidase; endopeptidase-24.11 reflects the changed morphology of Schwann cells while aminopeptidase-N, aminopeptidase-W and angiotensin converting enzyme become expressed by the actively phagocytosing macrophages.

Three markers of adult non-myelin-forming Schwann cells, 217c(Ran-1), A5E3 and GFAP: development and regulation by neuron–Schwann cell interactions

Immunohistochemical methods are used to investigate in detail the development and regulation of three proteins (217c(Ran-1), A5E3 and GFAP) specifically associated with adult non-myelin-forming Schwann cells in the rat sciatic nerve, from embryo day 15 to maturity. 217c(Ran-1), which is probably the NGF-receptor, and A5E3 are expressed by the majority of cells in the nerve at embryo day 15 and by essentially all cells at embryo day 18. GFAP first appears at embryo day 18; this is an intrinsically programmed developmental event which occurs in cultured Schwann cells even in the absence of serum. Postnatally, the expression of 217c(Ran-1), A5E3 and GFAP is suppressed in cells that form myelin but retained in non-myelin-forming Schwann cells. Mature myelin-forming cells nevertheless maintain the potential to express all three proteins but will only do so if removed from contact with myelinated axons. In neuron-free cultures Schwann cells express all three proteins. This work, together with our previous observations on N-CAM, shows that removal of a diverse set of surface proteins and a change in intermediate filament expression is one of the major consequences of axon to Schwann cell signalling during myelination in the rat sciatic nerve. Unlike myelin-forming cells, adult non-myelin-forming Schwann cells remain very similar to embryonic and newborn cells with respect to expression of surface proteins, in contrast to the previously established developmental changes that occur in their surface lipids.

Isolation of rat Schwann cell lines: Use of SV40 T antigen gene regulated by synthetic metallothionein promoters

Synthetic promoter elements from the mouse metallothionein-I promoter controlling the expression of SV40 T antigen have been tested for their efficacy in cloning rat Schwann cell lines that retained the characteristic properties of these cells and could be passed in culture indefinitely. The constructed promoters contained either four (MT 4) or five (MT 5) copies of a metal regulatory element 5′ to the CAAT and TATA elements from the HSV-1 thymidine kinase gene. Characterization of these promoters in transient expression assays and transformation assays showed that both MT 5 and MT 4 were approximately 10-fold and 15-fold, respectively, weaker than the wild-type MT-I promoter in the presence of heavy metal inducer. However, in the absence of inducer, the basal activity of both MT 5 and MT 4 was barely detectable and much lower than that of MT-I. Schwann cells were transfected with plasmids containing the SV40 T antigen gene under the control of the different metallothionein promoters and cell lines were established from each. Only with the MT 5 and MT 4 promoters were lines obtained that resembled secondary Schwann cells in culture in their morphology, generation time, and demonstration of contact inhibition. In the presence of zinc, the expression of T antigen in the lines derived with MT 5 and MT 4 was about 10-fold lower than that derived with MT-I. On removal of the inducer this level was reduced, and in one cell line T antigen was undetectable. Concomitant with the reduction in T antigen expression there was an increased expression of Po, a protein specific to myelin-forming Schwann cells, and a decreased expression of glial fibrillary acidic protein, a protein expressed only in nonmyelin-forming Schwann cells. These cell lines, therefore, closely resemble untransfected Schwann cells in culture.

Postnatal development of rat peripheral nerves: an immunohistochemical study of membrane lipids common to non-myelin forming Schwann cells, myelin forming Schwann cells and oligodendrocytes

Interest in the role of membrane lipids in Schwann cell function prompted this study of lipid antigens on myelin- and non-myelin forming Schwann cells. Using the monoclonal antibodies 07, which recognises galactocerebroside, 08, 09 and 011, the distribution and time course of expression of the 4 membrane lipids have been determined in Schwann cells of the rat sciatic nerve and sympathetic trunk, derived from 1-60-day-old rats. The proportion of Schwann cells binding each monoclonal antibody was found by dissociating the nerves and allowing 3 h for the cells to attach to coverslips, prior to double label immunofluorescence, using the monoclonal antibody in conjunction with antibodies to S100 as a general Schwann cell marker, or P 0 to distinguish cells which had formed myelin. All 4 lipid antigens were expressed by myelin forming Schwann cells, appearing just before, or at the time that the cells started to form myelin. Only 011 was restricted to myelin forming Schwann cells. Non-myelin forming Schwann cells expressed 07, 08 and 09. In the cervical sympathetic trunk, the developmental expression of these 3 lipids was essentially complete by postnatal day 20, whereas in the sciatic nerve, expression was not complete until days 40–60. The results show that the biochemical maturation in non-myelin forming Schwann cells differs greatly between different nerves, and may not be completed untill several weeks postnatally. The results also demonstrate that in addition to galactocerebroside, other similarities exist in the lipid composition of myelin and the plasma membrane of non-myelin forming Schwann cells since the lipids defined by 08 and 09 antibodies are found among both Schwann cell variants.

Nonmyelin-forming Schwann cells coexpress surface proteins and intermediate filaments not found in myelin-forming cells: a study of Ran-2, A5E3 antigen and glial fibrillary acidic protein

Little is known about the molecular properties of the nonmyelin-forming glia of the P.N.S. In addition, the relationship between these cells and C.N.S. glia is poorly understood, in contrast with the well-documented biochemical and functional similarities between myelin-forming Schwann cells and oligodendrocytes. We have shown elsewhere that two cell surface proteins found on astrocytes, Ran-2 and A5E3 antigen, and the intermediate filament protein, glial fibrillary acidic protein (GFAP), are all widely expressed in situ by nonmyelin-forming Schwann cells in various nerves from adult rats, but not by myelin-forming Schwann cells. Here we investigate whether these three proteins are exclusively coexpressed by the same population of Schwann cells in situ in adult nerves, or alternatively whether subpopulations of nonmyelin-forming Schwann cells can be defined that express one or two, but not all three of these antigens. The distribution of two surface proteins, Ran-2 and A5E3 antigen, both defined by monoclonal antibodies, is compared with the distribution of the intracellular intermediate filament protein GFAP in peripheral nerve trunks of adult rats using double label immunofluorescence. It is clear that the expression of all three proteins is confined to the same population of Schwann cells, namely, the Schwann cells surrounding the unmyelinated axons. The myelin-forming Schwann cells surrounding the larger axons do not express immunohistochemically detectable amounts of any of the proteins. In teased nerve preparations from the preganglionic sympathetic trunk, sciatic nerve, brachial plexus, dorsal and ventral roots, a complete correspondence is found between expression of Ran-2 and GFAP, and between A5E3 and GFAP in nonmyelin-forming Schwann cells. No Schwann cells positive for Ran-2 alone, A5E3 antigen alone, or GFAP alone were found. The same constellation of antigens is also expressed by astrocytes in the adult rat C.N.S. and enteric glia in the P.N.S. and not by other cell types. The phenotype A5E3+, Ran-2+, GFAP+ is thus characteristic of major categories of adult rat P.N.S. and C.N.S. glial cells, suggesting that this may reflect some common function for these three cell types.

Molecular identity, distribution and heterogeneity of glial fibrillary acidic protein: an immunoblotting and immunohistochemical study of Schwann cells, satellite cells, enteric glia and astrocytes.

Glial fibrillary acidic protein has been firmly established as the predominant component of astrocyte intermediate filaments. It has also been detected immunohistochemically in the glial cells of the enteric nervous system and some Schwann cells in the P.N.S. The molecular identity of this GFAP immunoreactivity in the P.N.S. has so far not been investigated. This study compares GFAP in the C.N.S. and P.N.S. of adult rats both immunochemically and immunohistochemically. Using SDS polyacrylamide gel electrophoresis combined with immunoblotting, and a polyclonal antiserum to brain GFAP, we show that the peripheral GFAP immunoreactivity resides in a polypeptide with a molecular weight of 49 kd, which is identical to that of rat brain GFAP. Furthermore, we find that this GFAP reactivity can be detected immunohistochemically in Schwann cells in a wide variety of nerves in the P.N.S. and in some satellite cells in both sensory and sympathetic ganglia, in addition to enteric glia. The pattern of distribution of GFAP filaments in Schwann cells suggests that, in the nerves surveyed, they may be expressed by most or all non-myelin forming Schwann cells but not by myelin-forming Schwann cells. We also show, using a monoclonal antibody to GFAP (anti-GFAP-3) in both immunohistochemical and immunoblotting studies, that the GFAP found in most peripheral glia is not identical to that of astrocytes since it lacks an antigenic determinant, defined by this monoclonal antibody, which is present in astrocytes. An exception to this finding is seen in the myenteric plexuses where immunohistochemically detectable GFAP is found in some, but not all, of the enteric glia, using the monoclonal antibody. Thus, the results suggest that GFA polypeptides may be a heterogeneous group, that share some common determinants and a common molecular weight, and show a widespread and complex distribution in the glia of both the C.N.S. and P.N.S.