Axonal regulation of Schwann cell proliferation and survival and the initial events of myelination requires PI 3-kinase activity.

In this report, we have investigated the signaling pathways that are activated by, and mediate the effects of, the neuregulins and axonal contact in Schwann cells. Phosphatidylinositol 3-kinase (PI 3-kinase) and mitogen-activated protein kinase kinase (MAPK kinase) are strongly activated in Schwann cells by glial growth factor (GGF), a soluble neuregulin, and by contact with neurite membranes; both kinase activities are also detected in Schwann cell-DRG neuron cocultures. Inhibition of the PI 3-kinase, but not the MAP kinase, pathway reversibly inhibited Schwann cell proliferation induced by GGF and neurites. Cultured Schwann cells undergo apoptosis after serum deprivation and can be rescued by GGF or contact with neurites; these survival effects were also blocked by inhibition of PI 3-kinase. Finally, we have examined the role of these signaling pathways in Schwann cell differentiation in cocultures. At early stages of coculture, inhibition of PI 3-kinase, but not MAPK kinase, blocked Schwann cell elongation and subsequent myelination but did not affect laminin deposition. Later, after Schwann cells established a one-to-one relationship with axons, inhibition of PI 3-kinase did not block myelin formation, but the myelin sheaths that formed were shorter, and the rate of myelin protein accumulation was markedly decreased. PI 3-kinase inhibition had no observable effect on the maintenance of myelin sheaths in mature myelinated cocultures. These results indicate that activation of PI 3-kinase by axonal factors, including the neuregulins, promotes Schwann cell proliferation and survival and implicate PI 3-kinase in the early events of myelination.     

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.     

The trunk neural crest and its early glial derivatives: a study of survival responses, developmental schedules and autocrine mechanisms

Regulation of survival during gliogenesis from the trunk neural crest is poorly understood. Using adapted survival assays, we directly compared crest cells and the crest-derived precursor populations that generate satellite cells and Schwann cells. A range of factors that supports Schwann cells and glial precursors does not rescue crest, with the major exception of neuregulin-1 that rescues crest cells provided they contact the extracellular matrix. Autocrine survival appears earlier in developing satellite cells than Schwann cells. Satellite cells also show early expression of S100beta, BFABP and fibronectin and early survival responses to IGF-1, NT-3 and PDGF-BB that in developing Schwann cells are not seen until the precursor/Schwann cell transition. These experiments define novel differences between crest cells and early glia and show that entry to the glial lineage is an important point for regulation of survival responses. They show that survival mechanisms among PNS glia differ early in development and that satellite cell development runs ahead of schedule compared to Schwann cells in several significant features.     

Platelet-derived growth factor-BB supports the survival of cultured rat Schwann cell precursors in synergy with neurotrophin-3

To understand the intimate axon-Schwann cell relationship required for the accurate development and regeneration of the peripheral nervous system (PNS), it is important to elucidate the repertoire of growth factors involved in this tightly regulated bi-directional dialogue. We focused on the identification and functional characterization of receptor tyrosine kinases (RTKs) in Schwann cells to gain insights into the corresponding growth factor ligands, which may be regulating the highly controlled differentiation of the Schwann cell lineage. Using an RT-PCR based differential display approach, we have identified 17 tyrosine kinases in embryonic rat sciatic nerves during the crucial transition from Schwann cell precursors to early Schwann cells. In this study, we have examined the expression and function of TrkC and the platelet-derived growth factor (PDGF) receptors alpha and beta on Schwann cell precursor cells. These receptors are expressed on freshly isolated Schwann cell precursors, and we show that PDGF-BB is able to rescue a subpopulation of these cells from apoptotic cell death in vitro. Furthermore, the TrkC-ligand neurotrophin-3 (NT-3) can act synergistically to potentiate this effect. However, PDGF-BB and NT-3 do not induce Schwann cell precursor proliferation or differentiation. Our data are consistent with a model suggesting that a combination of growth factors that include PDGF-BB and NT-3 are acting in concert and in synergy to regulate early Schwann cell development.     

SpL201: a conditionally immortalized Schwann cell precursor line that generates myelin

Dramatic progress has been made over recent years toward the elucidation of the mechanisms regulating lineage determination and cell survival in the developing peripheral nervous system. However, our understanding of Schwann cell development is limited. This is partly due to the difficulties in culturing primary Schwann cell precursor cells, the earliest developmental stage of the Schwann cell lineage defined to date. Both the inability to maintain cultured Schwann cell precursor cells in an undifferentiated state and the technical difficulties involved in their isolation have hampered progress. We have conditionally immortalized rat Schwann cell precursor cells using a retrovirally encoded EGFR/neu fusion protein to circumvent these problems and to generate a source of homogeneous cells. The resulting SpL201 cell line expresses p75 and nestin, two proteins expressed by neural crest-derived cells, as well as peripheral myelin protein 22, protein zero, and Oct-6 as markers of the Schwann cell lineage. When cultured in EGF-containing medium, the SpL201 cells proliferate and maintain an undifferentiated, Schwann cell precursor cell-like state. The cell line is dependent on EGF for survival but can differentiate into early Schwann cell-like cells in response to exogenous factors. Like primary rat Schwann cells, SpL201 cells upregulate Oct-6 and myelin gene expression in response to forskolin treatment. Furthermore, the SpL201 cell line can form myelin in the presence of axons in vitro and is capable of extensively remyelinating a CNS white matter lesion in vivo. Thus, this cell line provides a valuable and unique tool to study the Schwann cell lineage, including differentiation from the Schwann cell precursor cell stage through to myelination.     

Enteric neuroblasts require the phosphatidylinositol 3-kinase/Akt/Forkhead pathway for GDNF-stimulated survival

Glial cell line-derived neurotrophic factor (GDNF)/Ret signaling is required for enteric neural crest survival, proliferation, migration and process extension, but signaling pathways that mediate enteric nervous system (ENS) precursor development are poorly understood. We therefore examined GDNF effects on immunoselected ENS precursor survival and neuronal process extension in the presence of phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathway inhibitors. These studies demonstrated that GDNF promotes ENS precursor survival through phosphatidylinositol-3-kinase. Specifically, GDNF induces phosphorylation of Akt and loss of the Akt substrates FOXO1 and FOXO3a from the nucleus of ENS precursors. Furthermore, dominant negative Akt or active FOXO1 constructs promote ENS precursor cell death while a dominant negative FOXO1 construct prevents cell death. In contrast, the MAPK kinase inhibitor PD98059 did not influence ENS precursor survival or neurite extension. These data demonstrate a critical role for PI-3 kinase/Akt/FOXO signaling, but not for MAPK in ENS precursor survival and neurite extension.     

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.     

Mouse epidermal growth factor-responsive neural precursor cells increase the survival and functional capacity of embryonic rat dopamine neurons in vitro.

We have grown expanded populations of epidermal growth factor (EGF)-responsive mouse striatal precursor cells and subsequently co-cultured these with primary E14 rat ventral mesencephalon. The aim of these experiments was to induce dopaminergic (DA) neuronal phenotypes from the murine precursors. While no precursor cell-derived neurons were induced to express tyrosine hydroxylase (TH), there was a dramatic 30-fold increase in the survival of rat-derived TH-positive neurons in the co-cultures. The effect was not explicable solely in terms of total plating density, and was accompanied by a significantly enhanced capacity for [3H]dopamine uptake in the co-cultures compared to rat alone cultures. The present data show that, although primary rat E14 mesencephalic cells are incapable of inducing the development of DA neurons from EGF-responsive mouse neural precursor cells, such precursors will differentiate into cells capable of enhancing the survival and overall functional efficacy of primary embryonic dopamine neurons.     

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.     

Circulating Igf-I And Its Protective Role In The Pathogenesis Of Diabetic Angiopathy

Neuregulin-1 provides an important axonally derived signal for the survival and growth of developing Schwann cells, which is transmitted by the ErbB2/ ErbB3 receptor tyrosine kinases. Null mutations of the neuregulin-1, erbB2, or erbB3 mouse genes cause severe deficits in early Schwann cell development. Here, we employ Cre-loxP technology to introduce erbB2 mutations late in Schwann cell development, using a Krox20-cre allele. Cre-mediated erbB2 ablation occurs perinatally in peripheral nerves, but already at E11 within spinal roots. The mutant mice exhibit a widespread peripheral neuropathy characterized by abnormally thin myelin sheaths, containing fewer myelin wraps. In addition, in spinal roots the Schwann cell precursor pool is not correctly established. Thus, the Neuregulin signaling system functions during multiple stages of Schwann cell development and is essential for correct myelination. The thickness of the myelin sheath is determined by the axon diameter, and we suggest that trophic signals provided by the nerve determine the number of times a Schwann cell wraps an axon.     

Neuregulin signaling through a PI3K/Akt/Bad pathway in Schwann cell survival

beta-Neuregulin (betaNRG) is a potent Schwann cell survival factor that binds to and activates a heterodimeric ErbB2/ErbB3 receptor complex. We found that NRG receptor signaling rapidly activated phosphoinositide 3-kinase (PI3K) in serum-starved Schwann cells, while PI3K inhibitors markedly exacerbated apoptosis and completely blocked NRG-mediated rescue. NRG also rapidly signaled the phosphorylation of mitogen-activated protein kinase (MAPK) and the serine/threonine kinase Akt. The activation of Akt and MAPK in parallel pathways downstream from PI3K resulted in the phosphorylation of Bad at different serine residues. PI3K inhibitors that blocked NRG-mediated rescue also blocked the phosphorylation of Akt, MAPK, and Bad. However, selective inhibition of MEK-dependent Bad phosphorylation downstream from PI3K had no effect on NRG-mediated survival. Conversely, ectopic expression of wild-type Akt not only enhanced Bad phosphorylation but also enhanced autocrine- and NRG-mediated Schwann cell survival. Taken together, these results demonstrate that NRG receptor signaling through a PI3K/Akt/Bad pathway functions in Schwann cell survival.     

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 P_o, and in individual cells DNA synthesis is incompatible with the expression of P_o protein. Non-myelin-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.     

Pathways of migration of transplanted Schwann cells in the demyelinated mouse spinal cord

We have studied the behavior of Schwann cells transplanted at a distance from an induced myelin lesion of the adult mouse spinal cord. These transplanted cells were mouse Schwann cells arising from an immortalized cell line (MSC80) which expresses several Schwann cell phenotypes including the ability to produce myelin. The behavior of MSC80 cells was compared to that of purified rat Schwann cells transplanted in the same conditions. Schwann cells were labeled in vitro with the nuclear fluorochrome Hoechst 33342 and were transplanted at distances of 2-8 mm from a lysolecithin-induced myelin lesion in the spinal cord of shiverer and normal mice. Our results show that transplanted MSC80 cells migrated toward the lesion, in both shiverer and normal mouse spinal cord, preferentially along the ependyma, meninges, and blood vessels. They also migrated along white matter tracts but traveled a longer distance in shiverer (8 mm) than in normal (2-3 mm) white matter. Using these different pathways, MSC80 cells arrived within the lesion of shiverer and normal mouse spinal cord at the average speed of 166 p, m/hr (8 mm/48 hr). Migration was most efficient along the ependyma and the meninges where it attained up to 250 μm/hr. Migration was much slower in white matter tracts (95 μm/hr ± 54 in the shiverer and only 38 pm/hr ± 3 in the normal mouse). We also provide evidence for the specific attraction of MSC80 cells by the lysolecithin-induced lesion since (1) their number increased progressively with time in the lesion, and (2) MSC80 cells left their preferential pathways of migration specifically at the level of the lesion. Finally, combining the Hoechst Schwann cell labeling method with the immunohistochemical detection of the peripheral myelin protein, PO, we show that some of the MSC80 cells which have reached the lesion participate in myelin repair in both shiverer and normal lesioned mouse spinal cord. A series of control experiments performed with rat Schwann cells indicate that the migrating behavior of transplanted MSC80 cells was identical to that of purified but non-immortalized rat Schwann cells. © 1993 Wiley-Liss, Inc.