Fibroblast Growth Factors and Insulin Growth Factors Combine to Promote Survival of Rat Schwann Cell Precursors Without Induction of DNA Synthesis

In embryonic rat nerves, we recently identified an early cell in the Schwann cell lineage, the Schwann cell precursor. We found that when these cells were removed from contact with axons they underwent rapid apoptotic death, and that in a proportion of the cells this death could be prevented by basic fibroblast growth factor (bFGF, FGF-2). We now report that 100% of Schwann cell precursors isolated from peripheral nerves of 14-day-old-rat embryos can be rescued by a combination of insulin-like growth factor (IGF) 1 or 2 in combination with either acidic FGF (aFGF, FGF-1), bFGF or Kaposi's sarcoma FGF (K-FGF; FGF-4). The precursors display an absolute requirement for both an IGF and an FGF to achieve maximal survival. Elevation of intracellular levels of cAMP by forskolin does not result in a significant shift in the IGF/FGF dose-response curves. In contrast, the percentage of precursors rescued by FGF in the presence of insulin is dramatically increased by elevation of cAMP. These growth factor combinations did not stimulate DNA synthesis significantly in Schwann cell precursors. These findings show that cooperation between growth factors is required to suppress cell death in Schwann cell precursors, and suggest that survival and DNA synthesis are regulated by distinct growth factor combinations in these cells. The observations are consistent with the idea that survival regulation by FGFs and IGFs plays an important role in the development of glial cells in early embryonic nerves.     

Mesenchymal stem cells facilitate axon sorting, myelination, and functional recovery in paralyzed mice deficient in Schwann cell-derived laminin

Peripheral nerve function depends on a regulated process of axon and Schwann cell development. Schwann cells interact with peripheral neurons to sort and ensheath individual axons. Ablation of laminin γ1 in the peripheral nervous system (PNS) arrests Schwann cell development prior to radial sorting of axons. Peripheral nerves of laminin-deficient animals are disorganized and hypomyelinated. In this study, sciatic nerves of laminin-deficient mice were treated with syngenic murine adipose-derived stem cells (ADSCs). ADSCs expressed laminin in vitro and in vivo following transplant into mutant sciatic nerves. ADSC-treatment of mutant nerves caused endogenous Schwann cells to differentiate past the point of developmental arrest to sort and myelinate axons. This was shown by (1) functional, (2) ultrastructural, and (3) immunohistochemical analysis. Treatment of laminin-deficient nerves with either soluble laminin or the immortalized laminin-expressing cell line 3T3/L1 did not overcome endogenous Schwann cell developmental arrest. In summary, these results indicate that (1) laminin-deficient Schwann cells can be rescued, (2) a cell-based approach is beneficial in comparison with soluble protein treatment, and (3) mesenchymal stem cells modify sciatic nerve function via trophic effects rather than transdifferentiation in this system.     

Postnatal Schwann cell proliferation but not myelination is strictly and uniquely dependent on cyclin-dependent kinase 4 (cdk4)

Peripheral myelin formation depends on axonal signals that tightly control proliferation and differentiation of the associated Schwann cells. Here we demonstrate that the molecular program controlling proliferation of Schwann cells switches at birth. We have analyzed the requirements for three members of the cyclin-dependent kinase (cdk) family in Schwann cells using cdk-deficient mice. Mice lacking cdk4 showed a drastic decrease in the proliferation rate of Schwann cells at postnatal days 2 and 5, but proliferation was unaffected at embryonic day 18. In contrast, ablation of cdk2 and cdk6 had no significant influence on postnatal Schwann cell proliferation. Taken together, these findings indicate that postnatal Schwann cell proliferation is uniquely controlled by cdk4. Despite the lack of the postnatal wave of Schwann cell proliferation, axons were normally myelinated in adult cdk4-deficient sciatic nerves. Following nerve injury, Schwann cells lacking cdk4 were unable to re-enter the cell cycle, while Schwann cells deficient in cdk2 or cdk6 displayed proliferation rates comparable to controls. We did not observe compensatory effects such as elevated cdk4 levels in uninjured or injured nerves of cdk2 or cdk6-deficient mice. Our data demonstrate that prenatal and postnatal Schwann cell proliferation are driven by distinct molecular cues, and that postnatal proliferation is not a prerequisite for the generation of Schwann cell numbers adequate for correct myelination.     

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.     

Development of the Schwann cell lineage: from the neural crest to the myelinated nerve

The myelinating and nonmyelinating Schwann cells in peripheral nerves are derived from the neural crest, which is a transient and multipotent embryonic structure that also generates the other main glial subtypes of the peripheral nervous system (PNS). Schwann cell development occurs through a series of transitional embryonic and postnatal phases, which are tightly regulated by a number of signals. During the early embryonic phases, neural crest cells are specified to give rise to Schwann cell precursors, which represent the first transitional stage in the Schwann cell lineage, and these then generate the immature Schwann cells. At birth, the immature Schwann cells differentiate into either the myelinating or nonmyelinating Schwann cells that populate the mature nerve trunks. In this review, we will discuss the biology of the transitional stages in embryonic and early postnatal Schwann cell development, including the phenotypic differences between them and the recently identified signaling pathways, which control their differentiation and maintenance. In addition, the role and importance of the microenvironment in which glial differentiation takes place will be discussed.     

An in vivo analysis of Schwann cell programmed cell death in embryonic mice: the role of axons, glial growth factor, and the pro-apoptotic gene Bax

Building upon previous in vitro studies, the present investigation involves an in vivo examination of Schwann cell programmed cell death (PCD) and development in the brachial spinal ventral roots of embryonic mice. The period of Schwann cell PCD was found to occur between embryonic days (E) 11.5 and 18.5, which is in close coincidence with the PCD period of associated brachial motoneurons (E13.5-E18.5). Additionally, Schwann cells exhibited a peak in proliferation at E11.5, and differentiation from the precursor to the immature Schwann cell stage between E12.5 and E14.5. Axon-mediated Schwann cell survival was demonstrated in vivo by excitotoxic elimination of motoneurons and their axons, via NMDA treatment in utero. This treatment increased apoptotic Schwann cell death within degenerating ventral roots. Conversely, in utero co-treatment of glial growth factor (GGF) with NMDA resulted in decreased Schwann cell death, a finding which supports previous reports of the promotion of Schwann cell survival by GGF. Analysis of mice lacking Bax, a pro-apoptotic Bcl-2 protein, revealed that Schwann cell PCD occurred independently of Bax. However, owing to the lack of motoneuron PCD in Bax-knockout mice, and the corresponding increase in the number of ventral root axons, a decrease in Schwann cell PCD was observed during the normal period of motoneuron PCD. In conclusion, our findings regarding the regulation of Schwann cell development in vivo are consistent with the conclusions from in vitro studies, including a dependency on axons for survival and proliferation signals, timing of differentiation, and a dependency on GGF.     

The case for a central nervous system (CNS) origin for the Schwann cells that remyelinate CNS axons following concurrent loss of oligodendrocytes and astrocytes

In certain experimental and naturally occurring pathological situations in the central nervous system (CNS), demyelinated axons are remyelinated by Schwann cells. It has always been assumed that these Schwann cells are derived from Schwann cells associated with peripheral nerves. However, it has become apparent that CNS precursors can give rise to Schwann cells in vitro and following transplantation into astrocyte-free areas of demyelination in vivo. This paper compares the behaviour of remyelinating Schwann cells following transplantation of peripheral nerve derived Schwann cells over, and into, astrocyte-depleted areas of demyelination to that which follows transplantation of CNS cells and that seen in normally remyelinating ethidium bromide induced demyelinating lesions. It concludes that while the examination of normally remyelinating lesions can not resolve the origin of the remyelinating Schwann cells, the results from transplantation studies provide strong evidence that the Schwann cells that remyelinate CNS axons are most likely generated from CNS precursors. In addition these studies also indicate that the precursors that give rise to these Schwann cells are the same cells that give rise to remyelinating oligodendrocytes.     

Modulation of schwann cell phenotype by TGF-β1: Inhibition of P0 mRNA expression and downregulation of the low affinity NGF receptor

The phenotype of a fully differentiated, mature Schwann cell is appar-ently largely determined by Schwann cell-axon interactions. In vitro, elevation of intra-cellular cAMP levels in Schwann cells induces a phenotype which resembles that of a mature, i.e., axon-related, Schwann cell. Therefore, an important role for cAMP as a second messenger of axon-Schwann cell interactions in vivo is assumed. However, the effects of cAMP on Schwann cells are not restricted to induction of features of a mature phenotype. For example, elevation of intracellular cAMP levels results of also in a markedly increased synthesis of nerve growth factor (NGF) mRNA, which is barely detectable in intact sciatic nerves of adult animals. Furthermore, since cAMP induces myelin gene expression in cultured Schwann cells, additional regulatory mechanisms have to be postulated for the induction and maintenance of a mature non-myelinating Schwann cell phenotype. Here we show that a soluble protein “growth factor” can partially induce a non-myelinating mature Schwann cell phenotype in vitro. Treatment with transforming growth factor β1 (TGF-β1) results in a marked and rapid downregulation of the low affinty NGF receptor (NGFR) on cultured Schwann cells without induction of PO gene expression. In contrast, in agreement with previous studies, an increase in PO mRNA levels and a reduction in NGFR mRNA after cAMP elevation is much slower when compared with the effect of TGF-β1, suggesting the involvement of different intracellular mechanisms. Consistent with this hypothesis, we did not observe an induction of mRNA coding for TGR-β isoforms after cAMP elevation in cultured Schwann cells which constitutively synthesize TGF-β1 mRNA. © 1993 Wiley-Liss, Inc.     

Role of N-cadherin in Schwann cell precursors of growing nerves

In the present paper, we determine the localization and developmental regulation of N-cadherin in embryonic rat nerves and examine the role of N-cadherin in this system. We also identify a major transition in the architecture of embryonic nerves and relating it to N-cadherin expression. We find that in early embryonic nerves, N-cadherin is primarily expressed in Schwann cell precursors. Pronounced expression is seen at distal nerve fronts where these cells associate with growth cones, and the proximal nerve ends, in boundary cap cells. Unexpectedly, N-cadherin is downregulated as precursors generate Schwann cells, coinciding with the time at which most axons make target connections. Therefore, glial N-cadherin expression is essentially restricted to the period of axon outgrowth. We also provide evidence that N-cadherin supports the formation of contacts between Schwann cell precursors and show that these cells are a favorable substrate for axon growth, unlike N-cadherin-negative Schwann cells. Induction of N-cadherin expression in Schwann cells by neuregulin-1 restores their ability to form contacts and support axon growth. Finally, we show that the loss of glial N-cadherin during embryonic nerve development is accompanied by a transformation of nerve architecture, involving the appearance of endoneurial connective tissue space, fibroblasts, Schwann cell basal lamina, and blood vessels. Because N-cadherin is likely to promote the extensive glial contacts typical of the compact embryonic nerve, we suggest that N-cadherin loss at the time of Schwann cell generation allows endoneurial space to appear between the glial cells, a development that eventually permits the extensive interactions between connective tissue and individual axon-Schwann cell units necessary for myelination.     

P0 gene expression in Schwann cells is modulated by an increase of cAMP which is dependent on the presence of axons.

The role of cAMP in the regulation of P0 gene expression was investigated in Schwann cells of normal, regenerated, and permanently transected rat sciatic nerve. Forskolin treatment of endoneurial segments of rat sciatic nerve resulted in increased cAMP and P0 mRNA levels in normal and regenerated nerves but not in permanently transected nerves, where axonal regeneration is prevented. This increase of cAMP and P0 mRNA occurred within 30 and 90 min, respectively. P0 mRNA levels in the endoneurial segment of the permanently transected nerve were not increased with dibutyryl cAMP. The Schwann cells of the permanently transected nerve, however, retained the ability to myelinate 15 embryonic day (E15) dorsal root ganglia (DRG) neuron and neurite networks cultured in vitro. P0 mRNA levels increased within 4 days in transected endoneurium segments cocultured with E15 DRG neurons and neurites and further increased in 21 day myelinating cocultures. Although cAMP was not detectable in 4 day cocultures, it increased to detectable levels in 21 day cultures, suggesting that cAMP is involved in the myelinating process. These results indicate that the presence of the axon is required for the observed increase of cAMP and P0 mRNA levels and suggest that the increase of cAMP occurs within the axon which then presumably activates a different Schwann cell second messenger pathway to induce P0 gene expression.     

Chronically denervated rat schwann cells respond to GGF in vitro

C-erbB receptor/neuregulin signalling plays a significant role in Schwann cell function. In vivo, Schwann cells up-regulate expression of c-erbB receptors in the first month after injury, but receptor expression is down-regulated with time to levels that are not detectable immunohistochemically. The inability of chronically denervated Schwann cells to respond adequately to signals derived from regenerating axons may be one reason why delayed repair of an injured peripheral nerve frequently fails. We have examined the effects of GGF on denervated Schwann cells in vitro. A modified delayed dissociation technique was used to obtain adult rat Schwann cells from the distal stumps of transected sciatic nerves which had been acutely (7 days) or chronically (2–6 month) denervated. We found that in vitro denervated Schwann cells invariably expressed p75^NTR and c-erbB receptors. There was a progressive decrease in total cell yield and the percentage of cells with Schwann cell phenotype (p75^NTR and/S-100 or/laminin or /GFAP or/c-erbB positive); proliferation rate; migratory potential; and expression of the cell adhesion molecules N-CAM and N-cadherin, with increasing time of denervation. Addition of GGF2 had a significant stimulatory effect upon Schwann cell proliferation and migration, and an increased proportion of Schwann cells expressed N-CAM and N-cadherin, suggesting that these responses were mediated via GGF/c-erbB signalling. Our results support the view that it may be possible to manipulate chronically denervated Schwann cells so that they become more responsive to signals derived from regrowing axons. GLIA 24:290–303, 1998. © 1998 Wiley-Liss, Inc.     

Immature optic nerve glia of rat do not promote axonal regeneration when transplanted into a peripheral nerve

The ability of immature central nervous system (CNS) glia to promote axonal regeneration was studied by grafting segments of embryonic and neonatal rat optic nerves into the sciatic nerves of adult rats. Unexpectedly, very few axons regenerated through these grafts. The majority of the axons bypassed the grafts and were associated with Schwann cells. These results were similar to those obtained with grafts of adult rat optic nerves. The failure of immature CNS glia to promote axonal regeneration under these conditions suggests that they may be less effective than Schwann cells in promoting the regeneration and growth of axons.     

Schwann cell recruitment from intact nerve fibers

Two proximal branches of the rat facial nerve were transected and anastomosed end-to-end within a silicone tube, each of them being exposed to a massive invasion of ascending regenerating axons. The proximal nerves contained extremely large bundles of regenerated fibers, often associated with preexistent "parent fibers." The bundles showed many signs of rash and disordered cell proliferation and myelination. These included multiple Schwann cells surrounded by a common basement membrane, occurrence of different phases of myelination and even myelination of two axons by one Schwann cell. There was no evidence of mitogenic signals for fibrocytes. This model may be used for studying the mitogenic effect of axons on Schwann cells. It also suggests that so-called "groups of regenerating fibers" in neuropathy are caused by Schwann cell recruitment.     

The RNA-binding protein human antigen R controls global changes in gene expression during Schwann cell development

An important prerequisite to myelination in peripheral nerves is the establishment of one-to-one relationships between axons and Schwann cells. This patterning event depends on immature Schwann cell proliferation, apoptosis, and morphogenesis, which are governed by coordinated changes in gene expression. Here, we found that the RNA-binding protein human antigen R (HuR) was highly expressed in immature Schwann cells, where genome-wide identification of its target mRNAs in vivo in mouse sciatic nerves using ribonomics showed an enrichment of functionally related genes regulating these processes. HuR coordinately regulated expression of several genes to promote proliferation, apoptosis, and morphogenesis in rat Schwann cells, in response to NRG1, TGFβ, and laminins, three major signals implicated in this patterning event. Strikingly, HuR also binds to several mRNAs encoding myelination-related proteins but, contrary to its typical function, negatively regulated their expression, likely to prevent ectopic myelination during development. These functions of HuR correlated with its abundance and subcellular localization, which were regulated by different signals in Schwann cells.     

The effect of myelinating Schwann cells on axons

Myelinating Schwann cells control the number of neurofilaments and elevate the phosphorylation state of neurofilaments in the axon, eventually leading to the typical large axon caliber. Conversely, absence of myelin leads to lower amounts of neurofilaments, reduced phosphorylation levels, and smaller axon diameters. In addition, myelinating Schwann cells mediate the spacing of Na(+) channel clusters during development of the node of Ranvier. When axons are associated with mutant Schwann cells in inherited neuropathies, their calibers are reduced and their neurofilaments are less phosphorylated and more closely spaced. Also, axonal transport is reduced and axons degenerate at the distal ends of long nerves. Myelin-associated glycoprotein may mediate some aspects of Schwann cell-axon communication, but much remains to be learned about the molecular bases of Schwann cell-axon communication.     

Nearest-neighbor distance of intermediate filaments in axons and schwann cells

Summary To distinguish axons from Schwann cell processes in the denervated (Büngner's bands) and reinnervated peripheral nerves, the nearest-neighbor distance of intermediate filaments (NND) was measured in axons and Schwann cells from denervated and subsequent regenerating peripheral nerves. It was revealed that the NND was much larger in regenerating axons (41.9±14.1 nm) than in Schwann cell processes (23.1±7.1 nm in regeneration and 19.7±5.8 nm in denervation).In addition, the NND was also measured in the normal adult and developing peripheral nerves, and it became clear that in all cases the NND in axons (29.0–41.9 nm) was larger than in Schwann cells (19.7–23.1 nm). Thus, it can be generally considered that the NND is larger in axons than in Schwann cells. This fact can be used for the distinction between axons and Schwann cell processes, when the latter have a profile similar to that of the former as in Büngner's bands and in the regenerating nerves.     

Neurofibromatosis xenografts. Contribution to pathogenesis

We transplanted Schwann cells of 3 patients with neurofibromatosis from neurofibromas, sural nerve, and from a malignant schwannoma into sciatic nerves of immunoincompetent mice. Three and six months later, the grafts and distal nerve segments contained normal myelinated fibers. After rendering host animals immune competent again, neurofibroma and malignant schwannoma Schwann cells were rejected, but grafts retained normally myelinated fibers indicating that these were of mouse origin. Sural nerve Schwann cells from a neurofibromatosis patient were rejected also leaving naked axons in the grafted segments showing that human Schwann cells from the sural nerve of one patient had invested and myelinated the regenerating mouse axons. The nature of putative signals passing between axons and Schwann cells might be elucidated by the combination of human and animal cells in immunoincompetent host nerves. Hypothetical signals for myelination of mouse axons were normally received by sural nerve Schwann cells of a patient with neurofibromatosis, but not by Schwann cells from neurofibromas or malignant schwannomas.     

Three dimensional analysis of unmyelinated fibers in normal and pathologic autonomic nerves.

The technique of serial-section electron microscopy and diagrammatic three dimensional reconstructions has been used to assess normal and pathological unmyelinated nerve fibers from a peripheral autonomic nerve: the rat cervical sympathetic trunk. Within this predominantly unmyelinated nerve, there is a complex arrangement of axons into longitudinally oriented bundles brought together by chains of Schwann cells. Each bundle is subdivided into smaller components by the cytoplasmic processes of Schwann cells; such subdivisions, which are basal lamina-enclosed masses of Schwann cell cytoplasm, when viewed on cross-sectional electron micrographs, are termed Schwann cell units. The size and shape of each Schwann cell unit varies along the length of fibres, but the diameter of individual axons shows little variation over the segments studied. Axonal branching was not observed in normal unmyelinated nerves. Crush injury and x-irradiation produces different patterns of alteration in the axon-Schwann cell relationships of unmyelinated nerves. Following crush injury, Schwann cell processes increase in diameter and contain numerous small diameter axonal sprouts. Many of the regenerating axons remain thin while others reacquire a normal diameter. X-irradiation affects Schwann cells leading to retraction of their processes and the appearance of naked axonal segments.     

Axonal signals regulate the differentiation of non-myelin-forming Schwann cells: an immunohistochemical study of galactocerebroside in transected and regenerating nerves

Little is known about the factors involved in directing and maintaining the divergent differentiation of the 2 major Schwann cell variants, myelin and non-myelin-forming cells, in peripheral nerves. There is strong evidence that the differentiation of myelin-forming cells depends critically on cell-cell signaling through contact with appropriate axons. In this paper we ask whether this remarkable dependence of the Schwann cell on axonal contact for full differentiation is unique to those cells that form myelin or whether axonal signaling is also an important factor in the differentiation of non-myelin-forming Schwann cells. Sciatic nerves or cervical sympathetic trunks of adult rats were either transected or crushed and the axons allowed to degenerate and, in the case of crushed nerves, to regenerate into the distal stump for periods of time varying from 2 d to 9 weeks. The distal stump of the nerve was excised at specific times, the Schwann cells dissociated and immunolabeled with antibodies to galactocerebroside. In the sciatic nerve, which contains a mixture of non-myelin-forming and myelin-forming Schwann cells, transection resulted in a loss of galactocerebroside expression from the surface of all the Schwann cells in the distal stump over a 9 week period, irrespective of their original phenotype. In crushed sciatic nerves, where axons were allowed to regrow into the distal stumps, the number of Schwann cells expressing immunohistochemically detectable quantities of galactocerebroside in the stump declined over the first 3 weeks, but by 9 weeks after crush the total percentage of galactocerebroside-positive cells in the nerve had risen to control levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Schwann cell multiplication deficit in nerve roots of newborn dystrophic mice: A radioautographic and ultrastructural study

Neonatal Schwann cell development was studied in mice with genetic muscular dystrophy (dy^2J/dy^2J) to investigate the pathogenesis of the bare axonal segments which characterize certain spinal roots and peripheral nerves in these animals. From the day of birth to 3 weeks of age, dystrophic and control animals were injected with tritiated thymidine 1 hr prior to sacrifice. Cross-sections of their L4 ventral roots and sciatic nerves were processed for radioautography. Labelled and unlabelled Schwann cell nuclei, as well as numbers of myelinated fibres, were counted by phase microscopy and the same nerves examined by electron microscopy.On the day of birth, groups of large naked axons were present in the roots of dystrophic animals while in control roots most axons were isolated by Schwann cells or their processes. The number of myelinated fibres increased rapidly between days 1 and 9 in control roots but failed to increase in the dystrophic roots. On day 2, Schwann cell labelling indices were significantly lower in dystrophic roots than controls but were similar in dystrophic and control sciatic nerves. Many Schwann cells in the dystrophic roots did not encircle axons.It is concluded that the Schwann cell deficit in dystrophic mice is a developmental abnormality present at birth and associated with a neonatal impairment of Schwann cell multiplication. The presence of this unique abnormality of Schwann cell proliferation suggests an impairment of the mechanisms involved in the control of axon and Schwann cell populations during development.