The repair Schwann cell and its function in regenerating nerves.

Nerve injury triggers the conversion of myelin and non-myelin (Remak) Schwann cells to a cell phenotype specialized to promote repair. Distal to damage, these repair Schwann cells provide the necessary signals and spatial cues for the survival of injured neurons, axonal regeneration and target reinnervation. The conversion to repair Schwann cells involves de-differentiation together with alternative differentiation, or activation, a combination that is typical of cell type conversions often referred to as (direct or lineage) reprogramming. Thus, injury-induced Schwann cell reprogramming involves down-regulation of myelin genes combined with activation of a set of repair-supportive features, including up-regulation of trophic factors, elevation of cytokines as part of the innate immune response, myelin clearance by activation of myelin autophagy in Schwann cells and macrophage recruitment, and the formation of regeneration tracks, Bungner's bands, for directing axons to their targets. This repair programme is controlled transcriptionally by mechanisms involving the transcription factor c-Jun, which is rapidly up-regulated in Schwann cells after injury. In the absence of c-Jun, damage results in the formation of a dysfunctional repair cell, neuronal death and failure of functional recovery. c-Jun, although not required for Schwann cell development, is therefore central to the reprogramming of myelin and non-myelin (Remak) Schwann cells to repair cells after injury. In future, the signalling that specifies this cell requires further analysis so that pharmacological tools that boost and maintain the repair Schwann cell phenotype can be developed.

The effects of cAMP on differentiation of cultured Schwann cells: progression from an early phenotype (04+) to a myelin phenotype (P0+, GFAP-, N-CAM-, NGF-receptor-) depends on growth inhibition.

The present experiments were designed to clarify the relationship between cAMP elevation, proliferation and differentiation in Schwann cells. They were carried out on short-term cultures of cells obtained from neonatal rat sciatic nerves. It was found that the myelin-related phenotype was expressed in response to agents that elevate or mimic intracellular cAMP (forskolin, cholera toxin, cAMP analogues), provided cell division was absent. This phenotype included upregulation of the major myelin protein P0 and downregulation of GFAP, N-CAM, A5E3, and NGF receptor. In contrast, when cells were cultured in conditions where cell division occurred, elevation of intracellular cAMP produced an alternative response, characterized by DNA synthesis and absence of myelin-related differentiation. The cAMP mediated induction of an early Schwann cell antigen, 04, followed a different pattern since it was induced equally in dividing and nondividing cells. These observations are consistent with the proposal that during development of the rat sciatic nerve: (a) cAMP elevation, possibly induced by axon-associated factors, is a primary signal responsible for the induction of 04 expression in proliferating Schwann cells during the premyelination period; (b) subsequent withdrawal of cells associated with the larger axons from the cell cycle acts as a permissive secondary signal for induction of myelination, since in quiescent cells the ongoing cAMP elevation will trigger myelination.

Galactocerebroside is expressed by non-myelin-forming Schwann cells in situ.

Interest in the glycosphingolipid galactocerebroside (GC) is based on the consensus that in the nervous system it is expressed only by myelin-forming Schwann cells and oligodendrocytes, and that it has a specific role in the elaboration of myelin sheaths. We have investigated GC distribution in two rat nerves--the sciatic, containing a mixture of myelinated and non-myelinated axons, and the cervical sympathetic trunk, in which greater than 99% of axons are non-myelinated. Immunohistochemical experiments using mono- and polyclonal GC antibodies were carried out on teased nerves and cultured Schwann cells, and GC synthesis was assayed biochemically. Unexpectedly, we found that mature non-myelin-forming Schwann cells in situ and in short-term cultures express unambiguous GC immunoreactivity, comparable in intensity to that of myelinated fibers or myelin-forming cells in short-term cultures. GC synthesis was also detected in both sympathetic trunks and sciatic nerves. In the developing sympathetic trunk, GC was first seen at day 19 in utero, the number of GC-positive cells rising to approximately 95% at postnatal day 10. In contrast, the time course of GC appearance in the sciatic nerve shows two separate phases of increase, between day 18 in utero and postnatal day 1, and between postnatal days 20 and 35, at which stage approximately 94% of the cells express GC. These time courses suggest that Schwann cells, irrespective of subsequent differentiation pathway, start expressing GC at about the same time as cell division stops. We suggest that GC is a ubiquitous component of mature Schwann cell membranes in situ. Therefore, the role of GC needs to be reevaluated, since its function is clearly not restricted to events involved in myelination.

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.

The Schwann cell precursor and its fate: a study of cell death and differentiation during gliogenesis in rat embryonic nerves.

We have characterized a cell, the Schwann cell precursor, that represents a distinct intermediate differentiation stage in the process by which Schwann cells are generated from neural crest cells. The Schwann cell precursor shows radical differences from Schwann cells which include death regulation, antigenic phenotype, pattern of cell-cell interaction, migratory behavior, and morphology. In the nerves of the rat hind limb, Schwann cells are irreversibly generated from these during a brief period, essentially embryonic days 15-17. We also provide evidence that the survival of Schwann cell precursors is regulated by neurons and identify basic fibroblast growth factor as a potential key regulator of apoptosis in Schwann cell precursors and of precursor to Schwann cell conversion. These findings have implications for our understanding of gliogenesis in the peripheral nervous system.

Neu differentiation factor is a neuron-glia signal and regulates survival, proliferation, and maturation of rat Schwann cell precursors.

We show that beta forms of Neu differentiation factor (NDF), homologous to acetylcholine receptor-inducing activity, glial growth factor, and heregulin, prevent apoptotic death and stimulate DNA synthesis of the E14 Schwann cell precursor, an early cell in the rat Schwann cell lineage. When precursors are exposed to NDF in defined medium, they generate Schwann cells without the requirement for DNA synthesis and with a time course that is similar to that with which Schwann cells appear in embryonic nerves in vivo. Furthermore, a neuronal signal that also mediates precursor survival and maturation is blocked by the extracellular domain of the ErbB4 NDF receptor, a protein that specifically blocks the action of NDFs. These observations provide important evidence that NDF is one of the hitherto elusive neuron-glia signaling molecules long proposed to regulate development in the Schwann cell lineage.

Schwann cell-derived Desert hedgehog controls the development of peripheral nerve sheaths.

We show that Schwann cell-derived Desert hedgehog (Dhh) signals the formation of the connective tissue sheath around peripheral nerves. mRNAs for dhh and its receptor patched (ptc) are expressed in Schwann cells and perineural mesenchyme, respectively. In dhh-/- mice, epineurial collagen is reduced, while the perineurium is thin and disorganized, has patchy basal lamina, and fails to express connexin 43. Perineurial tight junctions are abnormal and allow the passage of proteins and neutrophils. In nerve fibroblasts, Dhh upregulates ptc and hedgehog-interacting protein (hip). These experiments reveal a novel developmental signaling pathway between glia and mesenchymal connective tissue and demonstrate its molecular identity in peripheral nerve. They also show that Schwann cell-derived signals can act as important regulators of nerve development.

Schwann cell precursors transplanted into the injured spinal cord multiply, integrate and are permissive for axon growth.

There is a strong current interest in the use of cell transplantation for the treatment of spinal cord injuries. We report here the novel and potentially useful properties of an early cell in the Schwann cell lineage, the Schwann cell precursor (SCP). The experiments reveal a striking difference between these cells and Schwann cells when transplanted into the CNS. Unlike Schwann cells, SCPs thrive in the CNS where they initially proliferate rapidly but then fall out of division, thus effectively filling up the large cystic cavities formed following crush injury, while avoiding tumor formation. By 8 weeks, SCPs had started to express S100beta protein, a marker that differentiates Schwann cells from SCPs and had formed an apparently stable, vascularized cell mass, which created a continuous cellular bridge across the cystic cavities. The formation of the surrounding glial scar was reduced by local spread of the transplanted cells into the surrounding CNS tissue, where the cells integrated intimately with astrocytes and attenuated the physical barrier they normally form. SCP transplantation also altered and reduced the expression of chondroitin sulfate proteoglycans around the injury site. Caudal to the SCP transplants there was a large increase in the number of axons, compared with that seen in nontransplanted control tissue, showing that the implants effectively support axonal growth or sprouting. SCPs have advantageous attributes for CNS repair, despite the fact that sticky tape removal and ladder crossing tests at 8 weeks did not reveal significant functional improvements when compared with control animals.

Schwann cell precursors: a favourable cell for myelin repair in the Central Nervous System.

Cell transplant therapies are currently under active consideration for a number of degenerative diseases. In the immune-mediated demyelinating-neurodegenerative disease multiple sclerosis (MS), only the myelin sheaths of the CNS are lost, while Schwann cell myelin of the PNS remains normal. This, and the finding that Schwann cells can myelinate CNS axons, has focussed interest on Schwann cell transplants to repair myelin in MS. However, the experimental use of these cells for myelin repair in animal models has revealed a number of problems relating to the incompatibility between peripheral glial cells and the CNS glial environment. Here, we have tested whether these difficulties can be avoided by using an earlier stage of the Schwann cell lineage, the Schwann cell precursor (SCP). For direct comparison of these two cell types, we implanted Schwann cells from post-natal rat nerves and SCPs from embryo day 14 (E14) rat nerves into the CNS under various experimental conditions. Examination 1 and 2 months later showed that in the presence of naked CNS axons, both types of cell form myelin that antigenically and ultrastructurally resembles that formed by Schwann cells in peripheral nerves. In terms of every other parameter we studied, however, the cells in these two implants behaved remarkably differently. As expected from previous work, Schwann cell implants survive poorly unless the cells find axons to myelinate, the cells do not migrate significantly from the implantation site, fail to integrate with host oligodendrocytes and astrocytes, and form little myelin when challenged with astrocyte-rich environment in the retina. Following SCP implantation, on the other hand, the cells survive well, migrate through normal CNS tissue, interface smoothly and intimately with host glial cells and myelinate extensively among the astrocytes of the retina. Furthermore, when implanted at a distance from a demyelinated lesion, SCPs but not Schwann cells migrate through normal CNS tissue to reach the lesion and generate new myelin. These features of SCP implants are all likely to be helpful attributes for a myelin repair cell. Since these cells also form Schwann cell myelin that is arguably likely to be resistant to MS pathology, they share some of the main advantages of Schwann cells without suffering from the disadvantages that render Schwann cells less than ideal candidates for transplantation into MS lesions.

Neural development. Fate diverted.

Signals that alter cell fate are probably crucial in metazoan development. Glial growth factor may play such a role in the mammalian neural crest, by regulating the generation of neurons and Schwann cells.

Schwann cells and their precursors emerge as major regulators of nerve development.

It is becoming ever clearer that Schwann cells and Schwann-cell precursors are an important source of developmental signals in embryonic and neonatal nerves. This article reviews experiments showing that these signals regulate the survival and differentiation of other cells in early nerves. The evidence indicates that glial-derived signals are necessary for neuronal survival at crucial periods of development, that they regulate the molecular and functional specialization of axons and that they control the maturation of the perineurial sheath that protects nerves from inflammation and unwanted macro-molecules produced in the surrounding tissues. Furthermore, an autocrine survival circuit enables Schwann cells in postnatal nerves to survive in the absence of axons, a vital requirement for successful nerve regeneration following injury. The molecular identity of these signals and their receptors is currently being determined.

Schwann cell development, differentiation and myelination.

Neu-differentiation factor (glial growth factor) has been established as an important regulator of early Schwann cell development, and the lineage relationship between immature Schwann cells and the neural crest has been clarified by the identification of the Schwann cell precursor. Progress has been made in identifying transcription factors that control Schwann cell development and in defining molecules that positively and negatively regulate myelin differentiation pathways. The tetraspan group has emerged as a set of proteins with prominent functions in Schwann cell biology.

Schwann cells: early lineage, regulation of proliferation and control of myelin formation.

This article reviews selected topics of particular relevance for understanding the process of Schwann cell development. It will discuss early commitment to the Schwann cell lineage and Schwann cell precursors, regulation of Schwann cell proliferation, and regulation of myelin formation.

Developing Schwann cells acquire the ability to survive without axons by establishing an autocrine circuit involving insulin-like growth factor, neurotrophin-3, and platelet-derived growth factor-BB.

Although Schwann cell precursors from early embryonic nerves die in the absence of axonal signals, Schwann cells in older nerves can survive in the absence of axons in the distal stump of transected nerves. This is crucially important, because successful axonal regrowth in a damaged nerve depends on interactions with living Schwann cells in the denervated distal stump. Here we show that Schwann cells acquire the ability to survive without axons by establishing an autocrine survival loop. This mechanism is absent in precursors. We show that insulin-like growth factor, neurotrophin-3, and platelet-derived growth factor-BB are important components of this autocrine survival signal. The secretion of these factors by Schwann cells has significant implications for cellular communication in developing nerves, in view of their known ability to regulate survival and differentiation of other cells including neurons.

Transforming growth factor beta (TGFbeta) mediates Schwann cell death in vitro and in vivo: examination of c-Jun activation, interactions with survival signals, and the relationship of TGFbeta-mediated death to Schwann cell differentiation.

In some situations, cell death in the nervous system is controlled by an interplay between survival factors and negative survival signals that actively induce apoptosis. The present work indicates that the survival of Schwann cells is regulated by such a dual mechanism involving the negative survival signal transforming growth factor beta (TGFbeta), a family of growth factors that is present in the Schwann cells themselves. We analyze the interactions between this putative autocrine death signal and previously defined paracrine and autocrine survival signals and show that expression of a dominant negative c-Jun inhibits TGFbeta-induced apoptosis. This and other findings pinpoint activation of c-Jun as a key downstream event in TGFbeta-induced Schwann cell death. The ability of TGFbeta to kill Schwann cells, like normal Schwann cell death in vivo, is under a strong developmental regulation, and we show that the decreasing ability of TGFbeta to kill older cells is attributable to a decreasing ability of TGFbeta to phosphorylate c-Jun in more differentiated cells.

The neurobiology of Schwann cells.

This selective review of Schwann cell biology focuses on questions relating to the origins, development and differentiation of Schwann cells and the signals that control these processes. The importance of neuregulins and their receptors in controlling Schwann cell precursor survival and generation of Schwann cells, and the role of these molecules in Schwann cell biology is addressed. The reciprocal signalling between peripheral glial cells and neurons in development and adult life revealed in recent years is highlighted, and the profound change in survival regulation from neuron-dependent Schwann cell precursors to adult Schwann cells that depend on autocrine survival signals is discussed. Besides providing neuronal and autocrine signals, Schwann cells signal to mesenchymal cells and influence the development of the connective tissue sheaths of peripheral nerves. The importance of Desert Hedgehog in this process is described. The control of gene expression during Schwann cell development and differentiation by transcription factors is reviewed. Knockout of Oct-6 and Krox-20 leads to delay or absence of myelination, and these results are related to morphological or physiological observations on knockout or mutation of myelin-related genes. Finally, the relationship between selected extracellular matrix components, integrins and the cytoskeleton is explored and related to disease.

Schwann Cells Secrete a PDGF-like Factor: Evidence for an Autocrine Growth Mechanism involving PDGF.

We have investigated the influence of platelet-derived growth factor (PDGF) in peripheral nervous system gliogenesis using two types of Schwann cell cultures. Short-term Schwann cell cultures grow very slowly, but when maintained in culture for several months the division rate of some cells increases, and cell lines can be established. We show that Schwann cells in both short- and long-term culture possess PDGF receptors and synthesize DNA in response to PDGF. Competitive binding experiments show that Schwann cells express mainly PDGF beta-receptors and respond better to PDGF-BB than to PDGF-AA. Conditioned media from short- and long-term Schwann cell cultures contain PDGF-like mitogenic activity, and anti-PDGF immunoglobin partially inhibits DNA synthesis in long-term Schwann cell cultures. Antibody neutralization experiments and Northern blot analyses both indicate that the predominant PDGF isoform in these cultures is PDGF-BB. PDGF-like activity is also detected in extracts of rat sciatic nerve. Taken together, these results suggest that PDGF-BB may stimulate Schwann cell proliferation in an autocrine manner during normal development.

Glial fibrillary acidic polypeptides in peripheral glia. Molecular weight, heterogeneity and distribution.

Glial fibrillary acidic (GFA) polypeptides are present in major categories of rat peripheral glia including non-myelin-forming Schwann cells, enteric glia and some satellite cells. They can be detected both immunochemically and immunohistochemically. The immunoreactivity is associated with a polypeptide which has an MW of 49 000, indistinguishable from that of glial fibrillary acidic protein (GFAP) from rat brain. In spite of this, the GFA polypeptides found in the peripheral nervous system and central nervous system are not identical since they can be distinguished both immunohistochemically and immunochemically by a monoclonal GFAP antibody which recognizes GFAP in astrocytes and some enteric glia, but not GFAP in non-myelin-forming Schwann cells, satellite cells and many enteric glia. GFA-related molecules can also be detected in human Schwann cells by immunofluorescence. The results suggest, however, that the glial filament polypeptides of peripheral glia and astrocytes are less closely related in the human than in the rat. The glial distribution of GFAP is closely paralleled by 2 cell surface proteins, Ran-2 and A5E3 antigen. Although GFAP, Ran-2 and A5E3 are individually expressed by diverse cell types, the phenotype GFAP+, Ran-2+, A5E3+ defines a narrow group including only non-myelin-forming Schwann cells, enteric glia and astrocytes. These observations suggest that the non-myelin-forming cells of the central and peripheral nervous system may share some common functions.

Rat Schwann cells produce interleukin-1.

There is increasing evidence that Schwann cells of peripheral nerves may be able to function as accessory cells, interacting with the immune system in T cell-mediated immune responses, by expression of the major histocompatibility complex (MHC) class II molecules. In addition to MHC class II-associated presentation of antigen to T lymphocytes, the release of a co-stimulatory factor, interleukin-1 (IL-1), is an essential function of accessory cells for T cell activation. In this study, we investigated if Schwann cells were able to produce IL-1. Purified cultures of neonatal and adult rat Schwann cells were incubated with various stimulatory agents. Supernatants and cell lysates were collected from these cultures and IL-1 activity was assayed. Both neonatal and adult rat Schwann cells produced IL-1 activity in response to bacterial antigens and the IL-1 activity was often higher in the cell lysate than in the supernatant. When stimulated neonatal or adult rat Schwann cells were examined with antibody against IL-1, strong immunolabelling was seen intracellularly, but no IL-1 was detected on the cell surface. Since IL-1 plays an important role in the initiation of immune responses, these observations support the view that Schwann cells may function as antigen-presenting cells, thereby taking part in neuroimmunological responses within peripheral nerves.

An immunohistochemical study of the distribution of enteric GABA-containing neurons in the rat and guinea-pig intestine.

gamma-Aminobutyric acid (GABA) antiserum was applied to sections of rat and guinea-pig intestine which were subsequently processed to reveal any immunoreactivity using either fluorescence or peroxidase techniques. Immunopositive fibres were demonstrated in stomach, duodenum, ileum and colon of rat and guinea-pig intestine. Myenteric ganglia and nerve bundles in the circular muscle contained immunopositive nerve fibres, while the longitudinal muscle, submucosa and mucosa were only rarely innervated. In favourable sections, immunopositive fibres could be seen running from the myenteric plexus into the circular muscle, thus suggesting that the GABA-immunopositive nerves in the circular muscle originate from neurons in the myenteric plexus. In both rat and guinea-pig, immunoreactive nerve cell bodies were most numerous in the myenteric plexus of the colon. In the rat, immunopositive fibres in the circular muscle were most abundant in the ileum, whereas in the guinea-pig it was the colon circular muscle that was most richly innervated. The results demonstrate that neurons which show GABA immunoreactivity are present along the length of the gastrointestinal tract. Their distribution in both myenteric ganglia and circular muscle is heterogeneous both within and between the two species studied. It is probable that this heterogeneity reflects the diversity and specificity of function of this class of enteric neurons.